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Sample records for microfluidic protein patterning

  1. Microfluidic Tools for Protein Crystallography

    NASA Astrophysics Data System (ADS)

    Abdallah, Bahige G.

    X-ray crystallography is the most widely used method to determine the structure of proteins, providing an understanding of their functions in all aspects of life to advance applications in fields such as drug development and renewable energy. New techniques, namely serial femtosecond crystallography (SFX), have unlocked the ability to unravel the structures of complex proteins with vital biological functions. A key step and major bottleneck of structure determination is protein crystallization, which is very arduous due to the complexity of proteins and their natural environments. Furthermore, crystal characteristics govern data quality, thus need to be optimized to attain the most accurate reconstruction of the protein structure. Crystal size is one such characteristic in which narrowed distributions with a small modal size can significantly reduce the amount of protein needed for SFX. A novel microfluidic sorting platform was developed to isolate viable ~200 nm -- ~600 nm photosystem I (PSI) membrane protein crystals from ~200 nm -- ~20 ?m crystal samples using dielectrophoresis, as confirmed by fluorescence microscopy, second-order nonlinear imaging of chiral crystals (SONICC), and dynamic light scattering. The platform was scaled-up to rapidly provide 100s of microliters of sorted crystals necessary for SFX, in which similar crystal size distributions were attained. Transmission electron microscopy was used to view the PSI crystal lattice, which remained well-ordered postsorting, and SFX diffraction data was obtained, confirming a high-quality, viable crystal sample. Simulations indicated sorted samples provided accurate, complete SFX datasets with 3500-fold less protein than unsorted samples. Microfluidic devices were also developed for versatile, rapid protein crystallization screening using nanovolumes of sample. Concentration gradients of protein and precipitant were generated to crystallize PSI, phycocyanin, and lysozyme using modified counterdiffusion

  2. Complex micropatterning of proteins within microfluidic channels.

    PubMed

    Kim, Miju; Doh, Junsang

    2014-01-01

    Microfluidic channels containing protein micropatterned surfaces are useful in many bioanalytical and biological applications. In this study, we developed a new method to integrate microfluidics and protein micropatterning by attaching poly(dimethylsiloxane) (PDMS) microfluidic channels to bio-friendly photoresist films via poly(dopamine) (PDA) adhesive. A bio-friendly photoresist poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP) was synthesized and used. By performing microscope projection photolithography (MPP) to the PDMP thin films within PDMS microchannels, complex micropatterns of proteins were successfully generated within microfluidic channels. PMID:25570075

  3. Patterned Plasmonic Nanoparticle Arrays for Microfluidic and Multiplexed Biological Assays.

    PubMed

    He, Jie; Boegli, Michelle; Bruzas, Ian; Lum, William; Sagle, Laura

    2015-11-17

    For applications ranging from medical diagnostics and drug screening to chemical and biological warfare detection, inexpensive, rapid-readout, portable devices are required. Localized surface plasmon resonance (LSPR) technologies show substantial promise toward meeting these goals, but the generation of portable, multiplexed and/or microfluidic devices incorporating sensitive nanoparticle arrays is only in its infancy. Herein, we have combined photolithography with Hole Mask Colloidal lithography to pattern uniform nanoparticle arrays for both microfluidic and multiplexed devices. The first proof-of-concept study is carried out with 5- and 7-channel microfluidic devices to acquire one-shot binding curves and protein binding kinetic data. The second proof-of-concept study involved the fabrication of a 96-spot plate that can be inserted into a standard plate reader for the multiplexed detection of protein binding. This versatile fabrication technique should prove useful in next generation chips for bioassays and genetic screening. PMID:26494412

  4. Rapid Protein Separations in Microfluidic Devices

    NASA Technical Reports Server (NTRS)

    Fan, Z. H.; Das, Champak; Xia, Zheng; Stoyanov, Alexander V.; Fredrickson, Carl K.

    2004-01-01

    This paper describes fabrication of glass and plastic microfluidic devices for protein separations. Although the long-term goal is to develop a microfluidic device for two-dimensional gel electrophoresis, this paper focuses on the first dimension-isoelectric focusing (IEF). A laser-induced fluorescence (LIF) imaging system has been built for imaging an entire channel in an IEF device. The whole-channel imaging eliminates the need to migrate focused protein bands, which is required if a single-point detector is used. Using the devices and the imaging system, we are able to perform IEF separations of proteins within minutes rather than hours in traditional bench-top instruments.

  5. Integrated Microfluidics for Protein Modification Discovery.

    PubMed

    Noach-Hirsh, Meirav; Nevenzal, Hadas; Glick, Yair; Chorni, Evelin; Avrahami, Dorit; Barbiro-Michaely, Efrat; Gerber, Doron; Tzur, Amit

    2015-10-01

    Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.

  6. Integrated Microfluidics for Protein Modification Discovery*

    PubMed Central

    Noach-Hirsh, Meirav; Nevenzal, Hadas; Glick, Yair; Chorni, Evelin; Avrahami, Dorit; Barbiro-Michaely, Efrat; Gerber, Doron; Tzur, Amit

    2015-01-01

    Protein post-translational modifications mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting post-translational modifications research, and both mass spectrometry and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple post-translational modifications analysis of freshly synthesized protein arrays (IMPA). The system's potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research. PMID:26276765

  7. Protein immobilization techniques for microfluidic assays

    PubMed Central

    Kim, Dohyun; Herr, Amy E.

    2013-01-01

    Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

  8. Microfluidic Mixers for Studying Protein Folding

    PubMed Central

    Waldauer, Steven A.; Wu, Ling; Yao, Shuhuai; Bakajin, Olgica; Lapidus, Lisa J.

    2012-01-01

    The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 μs2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4. Laminar flow is achieved for Reynolds numbers Re ≤100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 μm wide and 10 μm deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 μm in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 μL/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 μM (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM. The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 μm in 1 ms

  9. Multiplexed microfluidic quantification of proteins in serum

    NASA Astrophysics Data System (ADS)

    Rajan, Nitin; Rajauria, Sukumar; Cleland, Andrew

    2015-03-01

    Rapid and low cost immunoassays targeting proteins in blood or other bodily fluids are highly sought after for point-of-care devices and early screening of patients. Immunoturbidimetric assays utilize latex particles functionalized with antibodies, with particle aggregation in the presence of the analyte detected by a change in absorbance. Using a high throughput micro-fluidic particle analyzer based solely on electrical signals (resistive pulse sensing), we are able to accurately quantify the degree of aggregation by analyzing the changes in the particle size distribution. Thus we study the aggregation of streptavidin (SAv) coated beads in the presence of biotinylated bovine serum albumin as a proof-of-principle assay and extract the binding capacity of the SAv beads from the dose-response curve. We also use our aggregation measurement platform to characterize a commercial C-reactive protein (CRP) immunoturbidimetric assay (hsCRP, Diazyme Inc.). We obtain a linear calibration curve as well as a better limit of detection of CRP than that obtained by absorbance measurements. By using different bead sizes functionalized with different antibodies, multiplexed analyte detection is also possible. We demonstrate this by combining the commercial anti-CRP functionalized beads (0.4 microns) with biotin coated beads (1.0 microns), and carry out the simultaneous detection of SAv and CRP in a single sample.

  10. Patterned immobilization of antibodies within roll-to-roll hot embossed polymeric microfluidic channels.

    PubMed

    Feyssa, Belachew; Liedert, Christina; Kivimaki, Liisa; Johansson, Leena-Sisko; Jantunen, Heli; Hakalahti, Leena

    2013-01-01

    This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP). The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R(2) = 0.991) with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays.

  11. Patterned Immobilization of Antibodies within Roll-to-Roll Hot Embossed Polymeric Microfluidic Channels

    PubMed Central

    Feyssa, Belachew; Liedert, Christina; Kivimaki, Liisa; Johansson, Leena-Sisko; Jantunen, Heli; Hakalahti, Leena

    2013-01-01

    This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP). The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R2 = 0.991) with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays. PMID:23874811

  12. Patterned immobilization of antibodies within roll-to-roll hot embossed polymeric microfluidic channels.

    PubMed

    Feyssa, Belachew; Liedert, Christina; Kivimaki, Liisa; Johansson, Leena-Sisko; Jantunen, Heli; Hakalahti, Leena

    2013-01-01

    This paper describes a method for the patterned immobilization of capture antibodies into a microfluidic platform fabricated by roll-to-roll (R2R) hot embossing on poly (methyl methacrylate) (PMMA). Covalent attachment of antibodies was achieved by two sequential inkjet printing steps. First, a polyethyleneimine (PEI) layer was deposited onto oxygen plasma activated PMMA foil and further cross-linked with glutaraldehyde (GA) to provide an amine-reactive aldehyde surface (PEI-GA). This step was followed by a second deposition of antibody by overprinting on the PEI-GA patterned PMMA foil. The PEI polymer ink was first formulated to ensure stable drop formation in inkjet printing and the printed films were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). Anti-CRP antibody was patterned on PMMA foil by the developed method and bonded permanently with R2R hot embossed PMMA microchannels by solvent bonding lamination. The functionality of the immobilized antibody inside the microfluidic channel was evaluated by fluorescence-based sandwich immunoassay for detection of C-reactive protein (CRP). The antibody-antigen assay exhibited a good level of linearity over the range of 10 ng/ml to 500 ng/ml (R(2) = 0.991) with a calculated detection limit of 5.2 ng/ml. The developed patterning method is straightforward, rapid and provides a versatile approach for creating multiple protein patterns in a single microfluidic channel for multiplexed immunoassays. PMID:23874811

  13. Functional polymer sheet patterning using microfluidics.

    PubMed

    Li, Minggan; Humayun, Mouhita; Kozinski, Janusz A; Hwang, Dae Kun

    2014-07-22

    Poly(dimethylsiloxane) (PDMS)-based microfluidics provide a novel approach to advanced material synthesis. While PDMS has been successfully used in a wide range of industrial applications, due to the weak mechanical property channels generally possess low aspect ratios (AR) and thus produce microparticles with similarly low ARs. By increasing the channel width to nearly 1 cm, AR to 267, and implementing flow lithography, we were able to establish the slit-channel lithography. Not only does this allow us to synthesize sheet materials bearing multiscale features and tunable chemical anisotropy but it also allows us to fabricate functional layered sheet structures in a one-step, high-throughput fashion. We showcased the technique's potential role in various applications, such as the synthesis of planar material with micro- and nanoscale features, surface morphologies, construction of tubular and 3D layered hydrogel tissue scaffolds, and one-step formation of radio frequency identification (RFID) tags. The method introduced offers a novel route to functional sheet material synthesis and sheet system fabrication. PMID:24967616

  14. Protein Microarrays with Novel Microfluidic Methods: Current Advances

    PubMed Central

    Dixit, Chandra K.; Aguirre, Gerson R.

    2014-01-01

    Microfluidic-based micromosaic technology has allowed the pattering of recognition elements in restricted micrometer scale areas with high precision. This controlled patterning enabled the development of highly multiplexed arrays multiple analyte detection. This arraying technology was first introduced in the beginning of 2001 and holds tremendous potential to revolutionize microarray development and analyte detection. Later, several microfluidic methods were developed for microarray application. In this review we discuss these novel methods and approaches which leverage the property of microfluidic technologies to significantly improve various physical aspects of microarray technology, such as enhanced imprinting homogeneity, stability of the immobilized biomolecules, decreasing assay times, and reduction of the costs and of the bulky instrumentation.

  15. Multiplexing Ligand–Receptor Binding Measurements by Chemically Patterning Microfluidic Channels

    PubMed Central

    Shi, Jinjun; Yang, Tinglu; Cremer, Paul S.

    2012-01-01

    A method has been designed for patterning supported phospholipid bilayers (SLBs) on planar substrates and inside microfluidic channels. To do this, bovine serum albumin (BSA) monolayers were formed via adsorption at the liquid/solid interface. Next, this interfacial protein film was selectively patterned by using deep UV lithography. Subsequently, SLBs could be deposited in the patterned locations by vesicle fusion. By cycling through this process several times, spatially addressed bilayer arrays could be formed with intervening protein molecules serving as two-dimensional corrals. By employing this method, phospholipid bilayers containing various concentrations of ganglioside GM1 were addressed along the length of individual microfluidic channels. Therefore, the binding of GM1 with pentameric cholera toxin B (CTB) subunits could be probed. A seven-channel microfluidic device was fabricated for this purpose. Each channel was simultaneously patterned with four chemically distinct SLBs containing 0, 0.2, 0.5, and 2.0 mol % GM1, respectively. Varying concentrations of CTB were then introduced into each of the channels. With the use of total internal reflection fluorescence microscopy, it was possible to simultaneously abstract multiple equilibrium dissociation constants as a function of ligand density for the CTB-GM1 system in a single shot. PMID:18570383

  16. Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices.

    PubMed

    Kalish, Brent; Tsutsui, Hideaki

    2016-01-01

    We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive. PMID:27077551

  17. Hydrogel microfluidics for the patterning of pluripotent stem cells

    NASA Astrophysics Data System (ADS)

    Cosson, S.; Lutolf, M. P.

    2014-03-01

    Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate.

  18. RNA–protein binding kinetics in an automated microfluidic reactor

    PubMed Central

    Ridgeway, William K.; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R.

    2009-01-01

    Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome. PMID:19759214

  19. RNA-protein binding kinetics in an automated microfluidic reactor.

    PubMed

    Ridgeway, William K; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R

    2009-11-01

    Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA-protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic 'Riboreactor' has been designed and constructed to facilitate the study of kinetics of RNA-protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA-protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.

  20. Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics.

    PubMed

    Kipper, Sarit; Avrahami, Dorit; Bajorek, Monika; Gerber, Doron

    2016-01-01

    We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein-protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus-host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host. PMID:27464694

  1. Protein Microarrays with Novel Microfluidic Methods: Current Advances

    PubMed Central

    Dixit, Chandra K.; Aguirre, Gerson R.

    2014-01-01

    Microfluidic-based micromosaic technology has allowed the pattering of recognition elements in restricted micrometer scale areas with high precision. This controlled patterning enabled the development of highly multiplexed arrays multiple analyte detection. This arraying technology was first introduced in the beginning of 2001 and holds tremendous potential to revolutionize microarray development and analyte detection. Later, several microfluidic methods were developed for microarray application. In this review we discuss these novel methods and approaches which leverage the property of microfluidic technologies to significantly improve various physical aspects of microarray technology, such as enhanced imprinting homogeneity, stability of the immobilized biomolecules, decreasing assay times, and reduction of the costs and of the bulky instrumentation. PMID:27600343

  2. Patterning electrohydrodynamic flows with conductive obstacles in microfluidic channels.

    SciTech Connect

    Hill, Tyrone F.; Simmons, Blake Alexander; Barrett, Louis C.; Cummings, Eric B.; Fiechtner, Gregory J.; Harnett, Cindy K.; Skulan, Andrew J.

    2005-03-01

    Flow patterns with both recirculating and unidirectional characteristics are useful for controlled mixing and pumping within microfluidic devices. We have developed a fabrication process that converts injection-molded polymer chips into devices that demonstrate induced-charge electroosmosis (ICEO) effects (1,2) in AC fields. Polymeric insulating posts are coated with metal to produce a nonuniform zeta potential under an applied electric field. Induced flows are analyzed by particle image velocimetry. Stable, recirculating flow patterns are discussed, along with their potential to produce well-characterized and reversible streamlines for on-chip mixing in chemical separation and synthesis devices. Asymmetric conductive features can bias the flow direction, generating unidirectional pumping in an AC field. This pumping approach will be discussed in comparison with DC electrokinetic pumps we have studied.

  3. Protein Crystal Growth With the Aid of Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark

    2003-01-01

    Protein crystallography is one of three well-known methods to obtain the structure of proteins. A major rate limiting step in protein crystallography is protein crystal nucleation and growth, which is still largely a process conducted by trial-and-error methods. Many attempts have been made to improve protein crystal growth by performing growth in microgravity. Although the use of microgravity appears to improve crystal quality in some attempts, this method has been inefficient because several reasons: we lack a fundamental understanding of macromolecular crystal growth in general and of the influence of microgravity in particular, we have to start with crystal growth conditions in microgravity based on conditions on the ground and finally the hardware does not allow for experimental iteration without reloading samples on the ground. To partially accommodate the disadvantages of the current hardware, we have used microfluidic technology (Lab-on-a-Chip devices) to design the concept of a more efficient crystallization device, suitable for use on the International Space Station and in high-throughput applications on the ground. The concept and properties of microfluidics, the application design process, and the advances in protein crystal growth hardware will be discussed in this presentation. Some examples of proteins crystallized in the new hardware will be discussed, including the differences between conventional crystallization versus crystallization in microfluidics.

  4. Patterned Photonic Nitrocellulose for Pseudo-Paper Microfluidics.

    PubMed

    Gao, Bingbing; Liu, Hong; Gu, Zhongze

    2016-05-17

    We report a pseudo-paper microfluidic chip based on patterned photonic nitrocellulose. The photonic nitrocellulose is fabricated using self-assembled monodisperse SiO2 nanoparticles as template. The SiO2 nanoparticles form a photonic crystal having a close-packed hexagonal structure in the microchannels, so the resulting nitrocellulose has a complementary inverse-opal structure. After lamination, a hollow channel is obtained that is partially filled with the photonic nitrocellulose. Owing to the highly ordered photonic structure of the pseudo-paper chip, the flow profile of aqueous solution wicking through the channel is more uniform than conventional paper microfluidic chip. It is also found that the wicking rate of aqueous solution can be easily manipulated by changing the diameter of the self-assembled monodisperse SiO2 nanoparticles, which determines the pore size of the photonic nitrocellulose. The fluorescent enhancement property of the photonic nitrocellulose is used to increase the fluorescent intensity for multiplex detection of two cancer biomarkers. Label-free detection of human immunoglobin G based on the structure color of the photonic nitrocellulose is also demonstrated. PMID:27088587

  5. Wettability Control on Fluid-Fluid Displacements in Patterned Microfluidics

    NASA Astrophysics Data System (ADS)

    Zhao, B.; Trojer, M.; Cueto-Felgueroso, L.; Juanes, R.

    2014-12-01

    Two-phase flow in porous media is important in many natural and industrial processes like geologic CO2 sequestration, enhanced oil recovery, and water infiltration in soil. While it is well known that the wetting properties of porous media can vary drastically depending on the type of media and the pore fluids, the effect of wettability on fluid displacement continues to challenge our microscopic and macroscopic descriptions. Here we study this problem experimentally, starting with the classic experiment of two-phase flow in a capillary tube. We image the shape of the meniscus and measure the associated capillary pressure for a wide range of capillary numbers. We confirm that wettability exerts a fundamental control on meniscus deformation, and synthesize new observations on the dependence of the dynamic capillary pressure on wetting properties (contact angle) and flow conditions (viscosity contrast and capillary number). We compare our experiments to a macroscopic phase-field model of two-phase flow. We use the insights gained from the capillary tube experiments to explore the viscous fingering instability in the Hele-Shaw geometry in the partial-wetting regime. A key difference between a Hele-Shaw cell and a porous medium is the existence of micro-structures (i.e. pores and pore throats). To investigate how these micro-structrues impact fluid-fluid displacement, we conduct experiments on a planar microfluidic device patterned with vertical posts. We track the evolution of the fluid-fluid interface and elucidate the impact of wetting on the cooperative nature of fluid displacement during pore invasion events. We use the insights gained from the capillary tube and patterned microfluidics experiments to elucidate the effect of wetting properties on viscous fingering and capillary fingering in a Hele-Shaw cell filled with glass beads, where we observe a contact-angle-dependent stabilizing behavior for the emerging flow instabilities, as the system transitions from

  6. Modular microfluidics for point-of-care protein purifications

    SciTech Connect

    Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; Retterer, S. T.; Doktycz, M. J.

    2015-01-01

    Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.

  7. Microfluidic IEF technique for sequential phosphorylation analysis of protein kinases

    NASA Astrophysics Data System (ADS)

    Choi, Nakchul; Song, Simon; Choi, Hoseok; Lim, Bu-Taek; Kim, Young-Pil

    2015-11-01

    Sequential phosphorylation of protein kinases play the important role in signal transduction, protein regulation, and metabolism in living cells. The analysis of these phosphorylation cascades will provide new insights into their physiological functions in many biological functions. Unfortunately, the existing methods are limited to analyze the cascade activity. Therefore, we suggest a microfluidic isoelectric focusing technique (μIEF) for the analysis of the cascade activity. Using the technique, we show that the sequential phosphorylation of a peptide by two different kinases can be successfully detected on a microfluidic chip. In addition, the inhibition assay for kinase activity and the analysis on a real sample have also been conducted. The results indicate that μIEF is an excellent means for studies on phosphorylation cascade activity.

  8. Remote control of reversible localized protein adsorption in microfluidic devices.

    PubMed

    Hao, Nan; Li, Jin-Yi; Xiong, Meng; Xia, Xing-Hua; Xu, Jing-Juan; Chen, Hong-Yuan

    2014-08-13

    We present a facilely prepared graphene oxide (GO)/ poly(dimethylsiloxane) (PDMS) composite by dispersing nanosized GO in PDMS. On the basis of the combination of photothermal effects of GO and grafted thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), an optical-driving approach for remote control of localized wettability is realized. And this method has been successfully applied in the spatially controlled reversible protein adsorption in microfluidic devices. PMID:25068799

  9. Microfluidic-based patterning of embryonic stem cells for in vitro development studies.

    PubMed

    Suri, Shalu; Singh, Ankur; Nguyen, Anh H; Bratt-Leal, Andres M; McDevitt, Todd C; Lu, Hang

    2013-12-01

    In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments.

  10. Self-interaction chromatography of proteins on a microfluidic monolith

    PubMed Central

    Martin, Cristina; Lenhoff, Abraham M.

    2010-01-01

    A novel miniaturized system has been developed for measuring protein-protein interactions in solution with high efficiency and speed, and minimal use of protein. A chromatographic monolith synthesized in a capillary is used in the method to make interaction measurements by self-interaction chromatography (SIC) in a manner that, compared to column methods, is more efficient as well as more readily practicable even if only small amounts of protein are available. The microfluidic monolith requires much less protein for both column synthesis and the chromatographic measurements than a conventional SIC system, and in addition offers improved mass transfer and hence higher chromatographic efficiency than for previous SIC miniaturization systems. Protein self-interactions for catalase as a model protein, quantified by measurement of second virial coefficients, B22, were determined by SIC and follow trends that are consistent with previously reported values. Different column derivatization conditions were studied in order to optimize the chromatographic behavior of the microfluidic system for SIC measurements. Chromatographic sensitivity can be further increased by using different column synthesis conditions. PMID:21258647

  11. Wettability control on multiphase flow in patterned microfluidics.

    PubMed

    Zhao, Benzhong; MacMinn, Christopher W; Juanes, Ruben

    2016-09-13

    Multiphase flow in porous media is important in many natural and industrial processes, including geologic CO2 sequestration, enhanced oil recovery, and water infiltration into soil. Although it is well known that the wetting properties of porous media can vary drastically depending on the type of media and pore fluids, the effect of wettability on multiphase flow continues to challenge our microscopic and macroscopic descriptions. Here, we study the impact of wettability on viscously unfavorable fluid-fluid displacement in disordered media by means of high-resolution imaging in microfluidic flow cells patterned with vertical posts. By systematically varying the wettability of the flow cell over a wide range of contact angles, we find that increasing the substrate's affinity to the invading fluid results in more efficient displacement of the defending fluid up to a critical wetting transition, beyond which the trend is reversed. We identify the pore-scale mechanisms-cooperative pore filling (increasing displacement efficiency) and corner flow (decreasing displacement efficiency)-responsible for this macroscale behavior, and show that they rely on the inherent 3D nature of interfacial flows, even in quasi-2D media. Our results demonstrate the powerful control of wettability on multiphase flow in porous media, and show that the markedly different invasion protocols that emerge-from pore filling to postbridging-are determined by physical mechanisms that are missing from current pore-scale and continuum-scale descriptions. PMID:27559089

  12. Microsecond Microfluidic Mixing for Investigation of Protein Folding Kinetics

    SciTech Connect

    Hertzog, D E; Santiago, J G; Bakajin, O

    2003-06-25

    We have developed and characterized a mixer to study the reaction kinetics of protein folding on a microsecond timescale. The mixer uses hydrodynamic focusing of pressure-driven flow in a microfluidic channel to reduce diffusion times as first demonstrated by Knight et al.[1]. Features of the mixer include 1 {micro}s mixing times, sample consumptions of order 1 nl/s, loading sample volumes on the order of microliters, and the ability to manufacture in fused silica for compatibility with most spectroscopic methods.

  13. Microsecond Microfluidic Mixing for Investigation of Protein Folding Kinetics

    SciTech Connect

    Hertzog, D E; Santiago, J G; Bakajin, O

    2005-02-10

    We have developed and characterized a mixer to study the reaction kinetics of protein folding on a microsecond timescale. The mixer uses hydrodynamic focusing of pressure-driven flow in a microfluidic channel to reduce diffusion times as first demonstrated by Knight et al.[1]. Features of the mixer include 1 {micro}s mixing times, sample consumptions of order 1 nl/s, loading sample volumes on the order of microliters, and the ability to manufacture in fused silica for compatibility with most spectroscopic methods.

  14. Laminated microfluidic system for small sample protein analysis

    PubMed Central

    Saedinia, Sara; Nastiuk, Kent L.; Krolewski, John J.; Li, G. P.; Bachman, Mark

    2014-01-01

    We describe a technology based on lamination that allows for the production of highly integrated 3D devices suitable for performing a wide variety of microfluidic assays. This approach uses a suite of microfluidic coupons (“microfloupons”) that are intended to be stacked as needed to produce an assay of interest. Microfloupons may be manufactured in paper, plastic, gels, or other materials, in advance, by different manufacturers, then assembled by the assay designer as needed. To demonstrate this approach, we designed, assembled, and characterized a microfloupon device that performs sodium-dodecyl-sulfate polyacrylamide gel electrophoresis on a small sample of protein. This device allowed for the manipulation and transport of small amounts of protein sample, tight injection into a thin polyacrylamide gel, electrophoretic separation of the proteins into bands, and subsequent removal of the gel from the device for imaging and further analysis. The microfloupons are rugged enough to handle and can be easily aligned and laminated, allowing for a variety of different assays to be designed and configured by selecting appropriate microfloupons. This approach provides a convenient way to perform assays that have multiple steps, relieving the need to design highly sophisticated devices that incorporate all functions in a single unit, while still achieving the benefits of small sample size, automation, and high speed operation. PMID:24753728

  15. Laminated microfluidic system for small sample protein analysis.

    PubMed

    Saedinia, Sara; Nastiuk, Kent L; Krolewski, John J; Li, G P; Bachman, Mark

    2014-01-01

    We describe a technology based on lamination that allows for the production of highly integrated 3D devices suitable for performing a wide variety of microfluidic assays. This approach uses a suite of microfluidic coupons ("microfloupons") that are intended to be stacked as needed to produce an assay of interest. Microfloupons may be manufactured in paper, plastic, gels, or other materials, in advance, by different manufacturers, then assembled by the assay designer as needed. To demonstrate this approach, we designed, assembled, and characterized a microfloupon device that performs sodium-dodecyl-sulfate polyacrylamide gel electrophoresis on a small sample of protein. This device allowed for the manipulation and transport of small amounts of protein sample, tight injection into a thin polyacrylamide gel, electrophoretic separation of the proteins into bands, and subsequent removal of the gel from the device for imaging and further analysis. The microfloupons are rugged enough to handle and can be easily aligned and laminated, allowing for a variety of different assays to be designed and configured by selecting appropriate microfloupons. This approach provides a convenient way to perform assays that have multiple steps, relieving the need to design highly sophisticated devices that incorporate all functions in a single unit, while still achieving the benefits of small sample size, automation, and high speed operation.

  16. A microfluidic electrochemiluminescent device for detecting cancer biomarker proteins

    PubMed Central

    Sardesai, Naimish P.; Kadimisetty, Karteek; Faria, Ronaldo; Rusling, James F.

    2013-01-01

    We describe an electrochemiluminescence (ECL) immunoarray incorporated into a prototype microfluidic device for highly sensitive protein detection, and apply this system to accurate, sensitive measurements of prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum. The microfluidic system employed three molded polydimethylsiloxane (PDMS) channels on a conductive pyrolytic graphite chip (PG) (2.5 × 2.5 cm) inserted into a machined chamber and interfaced with a pump, switching valve and sample injector. Each of the three PDMS channels encompasses three 3 μL analytical wells. Capture-antibody-decorated single-wall carbon nanotube (SWCNT) forests are fabricated in the bottom of the wells. The antigen is captured by these antibodies on the well bottoms. Then a RuBPY-silica-secondary antibody (Ab2) label is injected to bind to antigen on the array, followed by injection of sacrificial reductant tripropylamine (TPrA) to produce ECL. For detection, the chip is placed into an open-top ECL measuring cell, and the channels are in contact with electrolyte in the chamber. Potential applied at 0.95 V vs. SCE oxidizes TPrA to produce ECL by redox cycling the RuBPY species in the particles, and ECL light is measured by a CCD camera. This approach achieved ultralow detection limits (DL) of 100 fg mL-1 for PSA (9 zeptomol) and 10 fg mL-1 (1 zeptomol) for IL-6 in calf serum, a 10-25 fold improvement of a similar non-microfluidic array. PSA and IL-6 in synthetic cancer patient serum samples were detected in 1.1 h and results correlated well with single-protein ELISAs. PMID:23307128

  17. Modular microfluidics for point-of-care protein purifications

    DOE PAGES

    Millet, L. J.; Lucheon, J. D.; Standaert, R. F.; Retterer, S. T.; Doktycz, M. J.

    2015-01-01

    Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured tomore » suit a variety of fluidic operations or biochemical processes. In conclusion, we demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules.« less

  18. Three-dimensional paper-based microfluidic device for assays of protein and glucose in urine.

    PubMed

    Sechi, Deidre; Greer, Brady; Johnson, Jesse; Hashemi, Nastaran

    2013-11-19

    The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.

  19. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

    PubMed Central

    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

    2016-01-01

    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput). PMID:26891303

  20. Microfluidic patterning of miniaturized DNA arrays on plastic substrates.

    PubMed

    Geissler, Matthias; Roy, Emmanuel; Diaz-Quijada, Gerardo A; Galas, Jean-Christophe; Veres, Teodor

    2009-07-01

    This paper describes the patterning of DNA arrays on plastic surfaces using an elastomeric, two-dimensional microcapillary system (muCS). Fluidic structures were realized through hot-embossing lithography using Versaflex CL30. Like elastomers based on poly(dimethylsiloxane), this thermoplastic block copolymer is able to seal a surface in a reversible manner, making it possible to confine DNA probes with a level of control that is unparalleled using standard microspotting techniques. We focus on muCSs that support arrays comprising up to 2 x 48 spots, each being 45 mum in diameter. Substrates were fabricated from two hard thermoplastic materials, poly(methylmethacrylate) and a polycyclic olefin (e.g., Zeonor 1060R), which were both activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride and N-hydroxysuccinimide to mediate covalent attachment of DNA molecules. The approach was exemplified by using 0.25-32 muM solutions of amino-modified oligonucleotides labeled with either Cy3 or Cy5 fluorescent dye in phosphate-buffered saline, allowing for a direct and sensitive characterization of the printed arrays. Solutions were incubated for durations of 1 to >48 h at 22, 30, and 40 degrees C to probe the conditions for obtaining uniform spots of high fluorescence intensity. The length (l) and depth (d) of microfluidic supply channels were both important with respect to depletion as well as evaporation of the solvent. While selective activation of the substrate proved helpful to limit unproductive loss of oligonucleotides along trajectories, incubation of solution in a humid environment was necessary to prevent uncontrolled drying of the liquid, keeping the immobilization process intact over extended periods of time. When combined, these strategies effectively promoted the formation of high-quality DNA arrays, making it possible to arrange multiple probes in parallel with a high degree of uniformity. Moreover, we show that resultant arrays are compatible

  1. X-ray transparent Microfluidics for Protein Crystallization and Biomineralization

    NASA Astrophysics Data System (ADS)

    Opathalage, Achini

    Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions. In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 A. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3.

  2. Low-cost, high-throughput fabrication of cloth-based microfluidic devices using a photolithographical patterning technique.

    PubMed

    Wu, Peijing; Zhang, Chunsun

    2015-03-21

    In this work, we first report a facile, low-cost and high-throughput method for photolithographical fabrication of microfluidic cloth-based analytical devices (μCADs) by simply using a cotton cloth as a substrate material and employing an inexpensive hydrophobic photoresist laboratory-formulated from commercially available reagents, which allows patterning of reproducible hydrophilic-hydrophobic features in the cloth with well-defined and uniform boundaries. Firstly, we evaluated the wicking properties of cotton cloths by testing the wicking rate in the cloth channel, in combination with scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) analyses. It is demonstrated that the wicking properties of the cloth microfluidic channel can be improved by soaking the cloth substrate in 20 wt% NaOH solution and by washing the cloth-based microfluidic patterns with 3 wt% SDS solution. Next, we studied the minimum dimensions achievable for the width of the hydrophobic barriers and hydrophilic channels. The results indicate that the smallest width for a desired hydrophobic barrier is designed to be 100 μm and that for a desired hydrophilic channel is designed to be 500 μm. Finally, the high-throughput μCADs prepared using the developed fabrication technique were demonstrated for colorimetric assays of glucose and protein in artificial urine samples. It has been shown that the photolithographically patterned μCADs have potential for a simple, quantitative colorimetric urine test. The combination of cheap cloth and inexpensive high-throughput photolithography enables the development of new types of low-cost cloth-based microfluidic devices, such as "microzone plates" and "gate arrays", which provide new methods to perform biochemical assays or control fluid flow. PMID:25656508

  3. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    PubMed

    Knowlton, Stephanie; Yu, Chu Hsiang; Ersoy, Fulya; Emadi, Sharareh; Khademhosseini, Ali; Tasoglu, Savas

    2016-06-01

    Three-dimensional (3D) printing offers potential to fabricate high-throughput and low-cost fabrication of microfluidic devices as a promising alternative to traditional techniques which enables efficient design iterations in the development stage. In this study, we demonstrate a single-step fabrication of a 3D transparent microfluidic chip using two alternative techniques: a stereolithography-based desktop 3D printer and a two-step fabrication using an industrial 3D printer based on polyjet technology. This method, compared to conventional fabrication using relatively expensive materials and labor-intensive processes, presents a low-cost, rapid prototyping technique to print functional 3D microfluidic chips. We enhance the capabilities of 3D-printed microfluidic devices by coupling 3D cell encapsulation and spatial patterning within photocrosslinkable gelatin methacryloyl (GelMA). The platform presented here serves as a 3D culture environment for long-term cell culture and growth. Furthermore, we have demonstrated the ability to print complex 3D microfluidic channels to create predictable and controllable fluid flow regimes. Here, we demonstrate the novel use of 3D-printed microfluidic chips as controllable 3D cell culture environments, advancing the applicability of 3D printing to engineering physiological systems for future applications in bioengineering.

  4. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs.

    PubMed

    Knowlton, Stephanie; Yu, Chu Hsiang; Ersoy, Fulya; Emadi, Sharareh; Khademhosseini, Ali; Tasoglu, Savas

    2016-06-01

    Three-dimensional (3D) printing offers potential to fabricate high-throughput and low-cost fabrication of microfluidic devices as a promising alternative to traditional techniques which enables efficient design iterations in the development stage. In this study, we demonstrate a single-step fabrication of a 3D transparent microfluidic chip using two alternative techniques: a stereolithography-based desktop 3D printer and a two-step fabrication using an industrial 3D printer based on polyjet technology. This method, compared to conventional fabrication using relatively expensive materials and labor-intensive processes, presents a low-cost, rapid prototyping technique to print functional 3D microfluidic chips. We enhance the capabilities of 3D-printed microfluidic devices by coupling 3D cell encapsulation and spatial patterning within photocrosslinkable gelatin methacryloyl (GelMA). The platform presented here serves as a 3D culture environment for long-term cell culture and growth. Furthermore, we have demonstrated the ability to print complex 3D microfluidic channels to create predictable and controllable fluid flow regimes. Here, we demonstrate the novel use of 3D-printed microfluidic chips as controllable 3D cell culture environments, advancing the applicability of 3D printing to engineering physiological systems for future applications in bioengineering. PMID:27321481

  5. Flexible microfluidic cloth-based analytical devices using a low-cost wax patterning technique.

    PubMed

    Nilghaz, Azadeh; Wicaksono, Dedy H B; Gustiono, Dwi; Abdul Majid, Fadzilah Adibah; Supriyanto, Eko; Abdul Kadir, Mohammed Rafiq

    2012-01-01

    This paper describes the fabrication of microfluidic cloth-based analytical devices (μCADs) using a simple wax patterning method on cotton cloth for performing colorimetric bioassays. Commercial cotton cloth fabric is proposed as a new inexpensive, lightweight, and flexible platform for fabricating two- (2D) and three-dimensional (3D) microfluidic systems. We demonstrated that the wicking property of the cotton microfluidic channel can be improved by scouring in soda ash (Na(2)CO(3)) solution which will remove the natural surface wax and expose the underlying texture of the cellulose fiber. After this treatment, we fabricated narrow hydrophilic channels with hydrophobic barriers made from patterned wax to define the 2D microfluidic devices. The designed pattern is carved on wax-impregnated paper, and subsequently transferred to attached cotton cloth by heat treatment. To further obtain 3D microfluidic devices having multiple layers of pattern, a single layer of wax patterned cloth can be folded along a predefined folding line and subsequently pressed using mechanical force. All the fabrication steps are simple and low cost since no special equipment is required. Diagnostic application of cloth-based devices is shown by the development of simple devices that wick and distribute microvolumes of simulated body fluids along the hydrophilic channels into reaction zones to react with analytical reagents. Colorimetric detection of bovine serum albumin (BSA) in artificial urine is carried out by direct visual observation of bromophenol blue (BPB) colour change in the reaction zones. Finally, we show the flexibility of the novel microfluidic platform by conducting a similar reaction in a bent pinned μCAD.

  6. Flexible microfluidic cloth-based analytical devices using a low-cost wax patterning technique.

    PubMed

    Nilghaz, Azadeh; Wicaksono, Dedy H B; Gustiono, Dwi; Abdul Majid, Fadzilah Adibah; Supriyanto, Eko; Abdul Kadir, Mohammed Rafiq

    2012-01-01

    This paper describes the fabrication of microfluidic cloth-based analytical devices (μCADs) using a simple wax patterning method on cotton cloth for performing colorimetric bioassays. Commercial cotton cloth fabric is proposed as a new inexpensive, lightweight, and flexible platform for fabricating two- (2D) and three-dimensional (3D) microfluidic systems. We demonstrated that the wicking property of the cotton microfluidic channel can be improved by scouring in soda ash (Na(2)CO(3)) solution which will remove the natural surface wax and expose the underlying texture of the cellulose fiber. After this treatment, we fabricated narrow hydrophilic channels with hydrophobic barriers made from patterned wax to define the 2D microfluidic devices. The designed pattern is carved on wax-impregnated paper, and subsequently transferred to attached cotton cloth by heat treatment. To further obtain 3D microfluidic devices having multiple layers of pattern, a single layer of wax patterned cloth can be folded along a predefined folding line and subsequently pressed using mechanical force. All the fabrication steps are simple and low cost since no special equipment is required. Diagnostic application of cloth-based devices is shown by the development of simple devices that wick and distribute microvolumes of simulated body fluids along the hydrophilic channels into reaction zones to react with analytical reagents. Colorimetric detection of bovine serum albumin (BSA) in artificial urine is carried out by direct visual observation of bromophenol blue (BPB) colour change in the reaction zones. Finally, we show the flexibility of the novel microfluidic platform by conducting a similar reaction in a bent pinned μCAD. PMID:22089026

  7. Identification of microfluidic two-phase flow patterns in lab-on-chip devices.

    PubMed

    Yang, Zhaochu; Dong, Tao; Halvorsen, Einar

    2014-01-01

    This work describes a capacitive sensor for identification of microfluidic two-phase flow in lab-on-chip devices. With interdigital electrodes and thin insulation layer utilized, this sensor is capable of being integrated with the microsystems easily. Transducing principle and design considerations are presented with respect to the microfluidic gas/liquid flow patterns. Numerical simulation results verify the operational principle. And the factors affecting the performance of the sensor are discussed. Besides, a feasible process flow for the fabrication is also proposed.

  8. Fabrication of microfluidic devices containing patterned microwell arrays.

    PubMed

    Henley, W Hampton; Dennis, Patty J; Ramsey, J Michael

    2012-02-01

    A rapid fabrication and prototyping technique to incorporate microwell arrays with sub-10 μm features within a single layer of microfluidic circuitry is presented. Typically, the construction of devices that incorporate very small architecture within larger components has required the assembly of multiple elements to form a working device. Rapid, facile production of a working device using only a single layer of molded polydimethylsiloxane (PDMS) and a glass support substrate is achieved with the reported fabrication technique. A combination of conventional wet-chemical etching for larger (≥20 μm) microchannel features and focused ion beam (FIB) milling for smaller (≤10 μm) microwell features was used to fabricate a monolithic glass master mold. PDMS/glass hybrid chips were then produced using simple molding and oxygen plasma bonding methods. Microwell structures were loaded with 3 μm antibody-functionalized dye-encoded polystyrene spheres, and a sandwich immunoassay for common cytokines was performed to demonstrate proof-of-principle. Potential applications for this device include highly parallel multiplexed sandwich immunoassays, DNA/RNA hybridization analyses, and enzyme linked immunosorbent assay (ELISA). The fabrication technique described can be used for rapid prototyping of devices wherever submicrometer- to micrometer-sized features are incorporated into a microfluidic device.

  9. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

    NASA Astrophysics Data System (ADS)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

  10. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

    PubMed Central

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

    2011-01-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. PMID:21974603

  11. A microfluidic, high throughput protein crystal growth method for microgravity.

    PubMed

    Carruthers, Carl W; Gerdts, Cory; Johnson, Michael D; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  12. A Microfluidic, High Throughput Protein Crystal Growth Method for Microgravity

    PubMed Central

    Carruthers Jr, Carl W.; Gerdts, Cory; Johnson, Michael D.; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions’ microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 103 cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories. PMID:24278480

  13. A microfluidic, high throughput protein crystal growth method for microgravity.

    PubMed

    Carruthers, Carl W; Gerdts, Cory; Johnson, Michael D; Webb, Paul

    2013-01-01

    The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.

  14. A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion

    PubMed Central

    Hansen, Carl L.; Skordalakes, Emmanuel; Berger, James M.; Quake, Stephen R.

    2002-01-01

    Producing robust and scalable fluid metering in a microfluidic device is a challenging problem. We developed a scheme for metering fluids on the picoliter scale that is scalable to highly integrated parallel architectures and is independent of the properties of the working fluid. We demonstrated the power of this method by fabricating and testing a microfluidic chip for rapid screening of protein crystallization conditions, a major hurdle in structural biology efforts. The chip has 480 active valves and performs 144 parallel reactions, each of which uses only 10 nl of protein sample. The properties of microfluidic mixing allow an efficient kinetic trajectory for crystallization, and the microfluidic device outperforms conventional techniques by detecting more crystallization conditions while using 2 orders of magnitude less protein sample. We demonstrate that diffraction-quality crystals may be grown and harvested from such nanoliter-volume reactions. PMID:12486223

  15. Multiplexed microfluidic blotting of proteins and nucleic acids by parallel, serpentine microchannels.

    PubMed

    He, Sha; Zhang, Yi; Wang, Pei; Xu, Xingzhi; Zhu, Kui; Pan, Wenying; Liu, Wenwen; Cai, Kaiyong; Sun, Jiashu; Zhang, Wei; Jiang, Xingyu

    2015-01-01

    This work develops a high-throughput, high-efficiency and straightforward microfluidic blotting method for analyzing proteins and nucleic acids. Sample solutions containing antibodies (for protein detection) or hybridization probes (for nucleic acid detection) are introduced into the parallel, serpentine microchannels to specifically recognize the immobilized targets on the substrate, achieving the identification of multiple targets in multiple samples simultaneously. The loading control, molecular weight markers, and antigen/antibody titration are designed and integrated into the microfluidic chip, thus allowing for the quantification of proteins and nucleic acids. Importantly, we could easily distinguish the adjacent blotting bands inside parallel microchannels, which may be difficult to achieve in conventional blotting. The small dimensions of microfluidic channels also help to reduce the amount of probing molecules and to accelerate the biochemical reaction. Our microfluidic blotting could bypass the steps of blocking and washing, further reducing the operation time and complexity.

  16. Pattern Formation without Patterning Proteins in Cyanobacteria

    NASA Astrophysics Data System (ADS)

    Allard, Jun; Rutenberg, Andrew

    2006-03-01

    Filaments of cyanobacteria respond to nitrogen starvation by differentiating one cell in ten into a heterocyst, which is devoted to fixing atmospheric nitrogen. This is an example of self-organized pattern formation. We present a dynamical model explaining the initial selection of heterocysts in mutated cyanobacteria that are effectively without normal patterning proteins. Our simulations of this model produce distributions of heterocyst spacings that are consistent with experimental data, and lead to new qualitative predictions on the mechanisms of pattern formation in filamentous cyanobacteria. We discuss possible experimental tests of our results.

  17. Thermoplastic microfluidic devices and their applications in protein and DNA analysis

    PubMed Central

    Liu, Ke; Fan, Z. Hugh

    2013-01-01

    Microfluidics is a platform technology that has been used for genomics, proteomics, chemical synthesis, environment monitoring, cellular studies, and other applications. The fabrication materials of microfluidic devices have traditionally included silicon and glass, but plastics have gained increasing attention in the past few years. We focus this review on thermoplastic microfluidic devices and their applications in protein and DNA analysis. We outline the device design and fabrication methods, followed by discussion on the strategies of surface treatment. We then concentrate on several significant advancements in applying thermoplastic microfluidic devices to protein separation, immunoassays, and DNA analysis. Comparison among numerous efforts, as well as the discussion on the challenges and innovation associated with detection, is presented. PMID:21274478

  18. A novel approach to determine the efficacy of patterned surfaces for biofouling control in relation to its microfluidic environment.

    PubMed

    Halder, Partha; Nasabi, Mahyar; Lopez, Francisco Javier Tovar; Jayasuriya, Niranjali; Bhattacharya, Satinath; Deighton, Margaret; Mitchell, Arnan; Bhuiyan, Muhammed Ali

    2013-01-01

    Biofouling, the unwanted growth of sessile microorganisms on submerged surfaces, presents a serious problem for underwater structures. While biofouling can be controlled to various degrees with different microstructure-based patterned surfaces, understanding of the underlying mechanism is still imprecise. Researchers have long speculated that microtopographies might influence near-surface microfluidic conditions, thus microhydrodynamically preventing the settlement of microorganisms. It is therefore very important to identify the microfluidic environment developed on patterned surfaces and its relation with the antifouling behaviour of those surfaces. This study considered the wall shear stress distribution pattern as a significant aspect of this microfluidic environment. In this study, patterned surfaces with microwell arrays were assessed experimentally with a real-time biofilm development monitoring system using a novel microchannel-based flow cell reactor. Finally, computational fluid dynamics simulations were carried out to show how the microfluidic conditions were affecting the initial settlement of microorganisms.

  19. A novel approach to determine the efficacy of patterned surfaces for biofouling control in relation to its microfluidic environment.

    PubMed

    Halder, Partha; Nasabi, Mahyar; Lopez, Francisco Javier Tovar; Jayasuriya, Niranjali; Bhattacharya, Satinath; Deighton, Margaret; Mitchell, Arnan; Bhuiyan, Muhammed Ali

    2013-01-01

    Biofouling, the unwanted growth of sessile microorganisms on submerged surfaces, presents a serious problem for underwater structures. While biofouling can be controlled to various degrees with different microstructure-based patterned surfaces, understanding of the underlying mechanism is still imprecise. Researchers have long speculated that microtopographies might influence near-surface microfluidic conditions, thus microhydrodynamically preventing the settlement of microorganisms. It is therefore very important to identify the microfluidic environment developed on patterned surfaces and its relation with the antifouling behaviour of those surfaces. This study considered the wall shear stress distribution pattern as a significant aspect of this microfluidic environment. In this study, patterned surfaces with microwell arrays were assessed experimentally with a real-time biofilm development monitoring system using a novel microchannel-based flow cell reactor. Finally, computational fluid dynamics simulations were carried out to show how the microfluidic conditions were affecting the initial settlement of microorganisms. PMID:23789960

  20. Tape transfer atomization patterning of liquid alloys for microfluidic stretchable wireless power transfer.

    PubMed

    Jeong, Seung Hee; Hjort, Klas; Wu, Zhigang

    2015-01-01

    Stretchable electronics offers unsurpassed mechanical compliance on complex or soft surfaces like the human skin and organs. To fully exploit this great advantage, an autonomous system with a self-powered energy source has been sought for. Here, we present a new technology to pattern liquid alloys on soft substrates, targeting at fabrication of a hybrid-integrated power source in microfluidic stretchable electronics. By atomized spraying of a liquid alloy onto a soft surface with a tape transferred adhesive mask, a universal fabrication process is provided for high quality patterns of liquid conductors in a meter scale. With the developed multilayer fabrication technique, a microfluidic stretchable wireless power transfer device with an integrated LED was demonstrated, which could survive cycling between 0% and 25% strain over 1,000 times. PMID:25673261

  1. Tape Transfer Atomization Patterning of Liquid Alloys for Microfluidic Stretchable Wireless Power Transfer

    NASA Astrophysics Data System (ADS)

    Jeong, Seung Hee; Hjort, Klas; Wu, Zhigang

    2015-02-01

    Stretchable electronics offers unsurpassed mechanical compliance on complex or soft surfaces like the human skin and organs. To fully exploit this great advantage, an autonomous system with a self-powered energy source has been sought for. Here, we present a new technology to pattern liquid alloys on soft substrates, targeting at fabrication of a hybrid-integrated power source in microfluidic stretchable electronics. By atomized spraying of a liquid alloy onto a soft surface with a tape transferred adhesive mask, a universal fabrication process is provided for high quality patterns of liquid conductors in a meter scale. With the developed multilayer fabrication technique, a microfluidic stretchable wireless power transfer device with an integrated LED was demonstrated, which could survive cycling between 0% and 25% strain over 1,000 times.

  2. Tape Transfer Atomization Patterning of Liquid Alloys for Microfluidic Stretchable Wireless Power Transfer

    PubMed Central

    Jeong, Seung Hee; Hjort, Klas; Wu, Zhigang

    2015-01-01

    Stretchable electronics offers unsurpassed mechanical compliance on complex or soft surfaces like the human skin and organs. To fully exploit this great advantage, an autonomous system with a self-powered energy source has been sought for. Here, we present a new technology to pattern liquid alloys on soft substrates, targeting at fabrication of a hybrid-integrated power source in microfluidic stretchable electronics. By atomized spraying of a liquid alloy onto a soft surface with a tape transferred adhesive mask, a universal fabrication process is provided for high quality patterns of liquid conductors in a meter scale. With the developed multilayer fabrication technique, a microfluidic stretchable wireless power transfer device with an integrated LED was demonstrated, which could survive cycling between 0% and 25% strain over 1,000 times. PMID:25673261

  3. Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe.

    PubMed

    Kashyap, Aditya; Cors, Julien F; Lovchik, Robert D; Kaigala, Govind V

    2016-01-01

    The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies. PMID:27685165

  4. Patterning, integration and characterisation of polymer optical oxygen sensors for microfluidic devices.

    PubMed

    Nock, Volker; Blaikie, Richard J; David, Tim

    2008-08-01

    This paper describes a process for the layer-by-layer fabrication and integration of luminescent dye-based optical oxygen sensors into microfluidic devices. Application of oxygen-sensitive platinum(ii) octaethylporphyrin ketone fluorescent dye dissolved in polystyrene onto glass substrates by spin-coating was studied. Soft lithography with polydimethylsiloxane (PDMS) stamps and reactive ion etching in oxygen plasma were used to produce sensor patterns with a minimum feature size of 25 microm. Sensors patterns were integrated into a PDMS microfluidic device by plasma bonding. No degradation of the sensor response as a result of the lithography and pattern-transfer processes was detected. Gaseous and dissolved oxygen (DO) detection was characterised using fluorescence microscopy. The intensity signal ratio of the sensor films was found to increase almost two-fold from 3.6 to 6.8 by reducing film thickness from 1.3 microm to 0.6 microm. Calibration of DO measurement showed linear Stern-Volmer behaviour that was constant for flow rates from 0.5 to 2 mL min(-1). The calibrated sensors were subsequently used to demonstrate laterally resolved detection of oxygen inside a microfluidic channel. The fabrication process provides a novel, easy to use method for the repeatable integration of optical oxygen sensors into cell-culture and lab-on-a-chip devices.

  5. Protein expression patterns of the yeast mating response.

    PubMed

    Yuan, Haiyu; Zhang, Rongfei; Shao, Bin; Wang, Xuan; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

    2016-06-13

    Microfluidics, in combination with time-lapse microscopy, is a transformative technology that significantly enhances our ability to monitor and probe biological processes in living cells. However, high-throughput microfluidic devices mostly require sophisticated preparatory and setup work and are thus hard to adopt by non-experts. In this work, we designed an easy-to-use microfluidic chip, which enables tracking of 48 GFP-tagged yeast strains, with each strain under two different stimulus conditions, in a single experiment. We used this technology to investigate the dynamic pattern of protein expression during the yeast mating differentiation response. High doses of pheromone induce cell cycle arrest and the shmoo morphology, whereas low doses of pheromone lead to elongation and chemotrophic growth. By systematically analyzing the protein dynamics of 156 pheromone-regulated genes, we identified groups of genes that are preferentially induced in response to low-dose pheromone (elongation during growth) or high-dose pheromone (shmoo formation and cell cycle arrest). The protein dynamics of these genes may provide insights into the mechanisms underlying the differentiation switch induced by different doses of pheromone. PMID:27177258

  6. Topographically-patterned porous membranes in a microfluidic device as an in vitro model of renal reabsorptive barriers

    PubMed Central

    Frohlich, Else M.; Alonso, José Luis; Borenstein, Jeffrey T.; Zhang, Xin; Arnaout, M. Amin

    2015-01-01

    Models of reabsorptive barriers require both a means to provide realistic physiologic cues to and quantify transport across a layer of cells forming the barrier. Here we have topographically-patterned porous membranes with several user-defined pattern types. To demonstrate the utility of the patterned membranes, we selected one type of pattern and applied it to a membrane to serve as a cell culture support in a microfluidic model of a renal reabsorptive barrier. The topographic cues in the model resemble physiological cues found in vivo while the porous structure allows quantification of transport across the cell layer. Sub-micron surface topography generated via hot-embossing onto a track-etched polycarbonate membrane, fully replicated topographical features and preserved porous architecture. Pore size and shape were analyzed with SEM and image analysis to determine the effect of hot embossing on pore morphology. The membrane was assembled into a bilayer microfluidic device and a human kidney proximal tubule epithelial cell line (HK-2) and primary renal proximal tubule epithelial cells (RPTEC) were cultured to confluency on the membrane. Immunofluorescent staining of both cell types revealed protein expression indicative of the formation of a reabsorptive barrier responsive to mechanical stimulation: ZO-1 (tight junction), paxillin (focal adhesions) and acetylated α-tubulin (primary cilia). HK-2 and RPTEC aligned in the direction of ridge/groove topography of the membrane in the device, evidence that the device has mechanical control over cell response. This topographically-patterned porous membrane provides an in vitro platform on which to model reabsorptive barriers with meaningful applications for understanding biological transport phenomenon, underlying disease mechanisms, and drug toxicity. PMID:23636129

  7. An integrated microfluidic biochemical detection system for protein analysis with magnetic bead-based sampling capabilities.

    PubMed

    Choi, Jin-Woo; Oh, Kwang W; Thomas, Jennifer H; Heineman, William R; Halsall, H Brian; Nevin, Joseph H; Helmicki, Arthur J; Henderson, H Thurman; Ahn, Chong H

    2002-02-01

    This paper presents the development and characterization of an integrated microfluidic biochemical detection system for fast and low-volume immunoassays using magnetic beads, which are used as both immobilization surfaces and bio-molecule carriers. Microfluidic components have been developed and integrated to construct a microfluidic biochemical detection system. Magnetic bead-based immunoassay, as a typical example of biochemical detection and analysis, has been successfully performed on the integrated microfluidic biochemical analysis system that includes a surface-mounted biofilter and electrochemical sensor on a glass microfluidic motherboard. Total time required for an immunoassay was less than 20 min including sample incubation time, and sample volume wasted was less than 50 microl during five repeated assays. Fast and low-volume biochemical analysis has been successfully achieved with the developed biofilter and immunosensor, which is integrated to the microfluidic system. Such a magnetic bead-based biochemical detection system, described in this paper, can be applied to protein analysis systems. PMID:15100857

  8. Hydrogel microfluidics for the patterning of pluripotent stem cells.

    PubMed

    Cosson, S; Lutolf, M P

    2014-01-01

    Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate. PMID:24662945

  9. SPH Based Optimization of Electrowetting-Driven Digital Microfluidics with Advanced Actuation Patterns

    NASA Astrophysics Data System (ADS)

    Weiß, Dennis; Greiner, Andreas; Lienemann, Jan; Korvink, Jan G.

    2013-12-01

    Fast and thorough mixing is a crucial operation of digital microfluidic devices, where discrete and small fluid portions are moved and processed. In this paper, we want to analyze and to optimize the mixing process by substituting conventional motion and superposing oscillatory and translational modes. An accurate multiphase smoothed particle hydrodynamics (SPH) discretization for incompressible flow is instantiated. Different harmonic excitation patterns for the solid-liquid surface energy are applied and their influence on droplet mode shapes, formation of eddies and the Shannon entropy of droplet fluid components are measured. We tailor enhanced actuation patterns which improve mixing grade and reduce mixing time.

  10. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    SciTech Connect

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Berger, James M.

    2015-04-01

    A microfluidic platform has been developed for the capture and X-ray analysis of protein microcrystals, affording a means to improve the efficiency of XFEL and synchrotron experiments. X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  11. Controlling the magnetic field distribution on the micrometer scale and generation of magnetic bead patterns for microfluidic applications.

    PubMed

    Yu, Xu; Feng, Xuan; Hu, Jun; Zhang, Zhi-Ling; Pang, Dai-Wen

    2011-04-19

    As is well known, controlling the local magnetic field distribution on the micrometer scale in a microfluidic chip is significant and has many applications in bioanalysis based on magnetic beads. However, it is a challenge to tailor the magnetic field introduced by external permanent magnets or electromagnets on the micrometer scale. Here, we demonstrated a simple approach to controlling the local magnetic field distribution on the micrometer scale in a microfluidic chip by nickel patterns encapsulated in a thin poly(dimethylsiloxane) (PDMS) film under the fluid channel. With the precisely controlled magnetic field, magnetic bead patterns were convenient to generate. Moreover, two kinds of fluorescent magnetic beads were patterned in the microfluidic channel, which demonstrated that it was possible to generate different functional magnetic bead patterns in situ, and could be used for the detection of multiple targets. In addition, this method was applied to generate cancer cell patterns.

  12. Microfluidics for the analysis of membrane proteins: how do we get there?

    PubMed

    Battle, Katrina N; Uba, Franklin I; Soper, Steven A

    2014-08-01

    The development of fully automated and high-throughput systems for proteomics is now in demand because of the need to generate new protein-based disease biomarkers. Unfortunately, it is difficult to identify protein biomarkers that are low abundant when in the presence of highly abundant proteins, especially in complex biological samples such as serum, cell lysates, and other biological fluids. Membrane proteins, which are in many cases of low abundance compared to the cytosolic proteins, have various functions and can provide insight into the state of a disease and serve as targets for new drugs making them attractive biomarker candidates. Traditionally, proteins are identified through the use of gel electrophoretic techniques, which are not always suitable for particular protein samples such as membrane proteins. Microfluidics offers the potential as a fully automated platform for the efficient and high-throughput analysis of complex samples, such as membrane proteins, and do so with performance metrics that exceed their bench-top counterparts. In recent years, there have been various improvements to microfluidics and their use for proteomic analysis as reported in the literature. Consequently, this review presents an overview of the traditional proteomic-processing pipelines for membrane proteins and insights into new technological developments with a focus on the applicability of microfluidics for the analysis of membrane proteins. Sample preparation techniques will be discussed in detail and novel interfacing strategies as it relates to MS will be highlighted. Lastly, some general conclusions and future perspectives are presented.

  13. Understanding wax screen-printing: a novel patterning process for microfluidic cloth-based analytical devices.

    PubMed

    Liu, Min; Zhang, Chunsun; Liu, Feifei

    2015-09-01

    In this work, we first introduce the fabrication of microfluidic cloth-based analytical devices (μCADs) using a wax screen-printing approach that is suitable for simple, inexpensive, rapid, low-energy-consumption and high-throughput preparation of cloth-based analytical devices. We have carried out a detailed study on the wax screen-printing of μCADs and have obtained some interesting results. Firstly, an analytical model is established for the spreading of molten wax in cloth. Secondly, a new wax screen-printing process has been proposed for fabricating μCADs, where the melting of wax into the cloth is much faster (∼5 s) and the heating temperature is much lower (75 °C). Thirdly, the experimental results show that the patterning effects of the proposed wax screen-printing method depend to a certain extent on types of screens, wax melting temperatures and melting time. Under optimized conditions, the minimum printing width of hydrophobic wax barrier and hydrophilic channel is 100 μm and 1.9 mm, respectively. Importantly, the developed analytical model is also well validated by these experiments. Fourthly, the μCADs fabricated by the presented wax screen-printing method are used to perform a proof-of-concept assay of glucose or protein in artificial urine with rapid high-throughput detection taking place on a 48-chamber cloth-based device and being performed by a visual readout. Overall, the developed cloth-based wax screen-printing and arrayed μCADs should provide a new research direction in the development of advanced sensor arrays for detection of a series of analytes relevant to many diverse applications. PMID:26388382

  14. Understanding wax screen-printing: a novel patterning process for microfluidic cloth-based analytical devices.

    PubMed

    Liu, Min; Zhang, Chunsun; Liu, Feifei

    2015-09-01

    In this work, we first introduce the fabrication of microfluidic cloth-based analytical devices (μCADs) using a wax screen-printing approach that is suitable for simple, inexpensive, rapid, low-energy-consumption and high-throughput preparation of cloth-based analytical devices. We have carried out a detailed study on the wax screen-printing of μCADs and have obtained some interesting results. Firstly, an analytical model is established for the spreading of molten wax in cloth. Secondly, a new wax screen-printing process has been proposed for fabricating μCADs, where the melting of wax into the cloth is much faster (∼5 s) and the heating temperature is much lower (75 °C). Thirdly, the experimental results show that the patterning effects of the proposed wax screen-printing method depend to a certain extent on types of screens, wax melting temperatures and melting time. Under optimized conditions, the minimum printing width of hydrophobic wax barrier and hydrophilic channel is 100 μm and 1.9 mm, respectively. Importantly, the developed analytical model is also well validated by these experiments. Fourthly, the μCADs fabricated by the presented wax screen-printing method are used to perform a proof-of-concept assay of glucose or protein in artificial urine with rapid high-throughput detection taking place on a 48-chamber cloth-based device and being performed by a visual readout. Overall, the developed cloth-based wax screen-printing and arrayed μCADs should provide a new research direction in the development of advanced sensor arrays for detection of a series of analytes relevant to many diverse applications.

  15. Fabrication of thermo-responsive microfluidic membrane using photopolymerization patterning

    NASA Astrophysics Data System (ADS)

    Kim, Hyejeong; Lee, Sang Joon

    2015-11-01

    The programmed manipulation of responsive functional hydrogels is receiving large attention because of its unique functions and wide range of engineering applications. In this study, we developed an innovative stomata-inspired membrane (SIM) by fabricating a temperature-responsive hydrogel with a simple, cost effective, and high-throughput photopolymerization patterning process. Polymerization-induced diffusion on the macro-scale surface gives rise to form a multi-parted polymer membrane with fine pores by simple UV irradiation. After heating the SIM, the less deformable thick frame supports the whole structure, and the highly deformable thin base regulates the size of pores. The morphological configuration of the SIM can be easily changed by varying the solution composition or selecting a suitable photomask with different pattern. The developed SIM has the special sensing-to-actuation functions of stimuli-responsive hydrogels. This membrane with temperature-responsive pores would be potentially utilized in numerous practical applications, such as filter membranes with self-adjustable pores, membrane-based sensors, membrane-based actuators, and multi-functional membranes etc. This study was supported by the National Research Foundation of Korea (NRF) and funded by the Korean government (MSIP) (Grant No. 2008-0061991).

  16. Microfabrication- and microfluidics-based patterning of cultured neuronal network.

    PubMed

    Takayama, Yuzo; Kotake, Naoki; Haga, Tatsuya; Suzuki, Takafumi; Mabuchi, Kunihiko

    2011-01-01

    The cultured neuronal monolayer has been a promising model system for studying the neuronal dynamics, from single cell to network-wide level. Randomness in the reconstituted network structure has, however, hindered regulated signal transmissions from one neuron to another or from one neuronal population to another. Applying microfabrication-based cell patterning techniques is a promising approach to handling these problems. In the present study, we attempt to regulate the direction of axon development and the pathway of signal transmissions in cultured neuronal networks using micro-fabrication and - fluidic techniques. We created a PDMS-based culture device, which consisted of arrays of U-shaped cell trapping microwells, and placed it onto a chemically micropatterned glass substrate. After 6 days in vitro, we confirmed that cortical neurons extended neurites along the medium flow direction and the micropatterned regions. PMID:22255121

  17. TiO2 coated microfluidic devices for recoverable hydrophilic and hydrophobic patterns

    NASA Astrophysics Data System (ADS)

    Lee, Jin-Hyung; Kim, Sang Kyung; Park, Hyung-Ho; Kim, Tae Song

    2015-03-01

    We report a simple method for modifying the surfaces of plastic microfluidic devices through dynamic coating process with a nano-colloidal TiO2 sol. The surface of the thermoplastic, cyclic olefin copolymer (COC) was coated with the TiO2 film, that displayed an effective photocatalytic property. The hydrophilic surface is obtained in the TiO2-coated zone of a microfluidic channel, and TiO2 coated surface degradation can be reversed easily by UV irradiation. The present work shows a photocatalytic activity concerning the effect of TiO2 coating density, which is controlled by the number of coating cycles. The hydrophilized surface was characterized by the contact angle of water and the TiO2 coated COC surface reduced the water contact angle from 85° to less than 10° upon UV irradiation. The photocatalytic effect of the layer that was coated five times with TiO2 was excellent, and the super-hydrophilicity of the TiO2 surface could be promptly recovered after 10 months of storage at atmospheric conditions. The COC microfluidic devices, in which TiO2 has been freshly deposited and aged for 10 months, were capable of generating water-in oil-in water (W/O/W) double emulsions easily and uniformly by simple control of the flow rates for demonstration of excellent hydrophilic patterning and recovery of the TiO2 coated in the microchannels.

  18. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells.

    PubMed

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S; Lu, Chang

    2016-01-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples. PMID:27388549

  19. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells

    PubMed Central

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S.; Lu, Chang

    2016-01-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples. PMID:27388549

  20. Paramagnetic Structures within a Microfluidic Channel for Enhanced Immunomagnetic Isolation and Surface Patterning of Cells

    NASA Astrophysics Data System (ADS)

    Sun, Chen; Hassanisaber, Hamid; Yu, Richard; Ma, Sai; Verbridge, Scott S.; Lu, Chang

    2016-07-01

    In this report, we demonstrate a unique method for embedding magnetic structures inside a microfluidic channel for cell isolation. We used a molding process to fabricate these structures out of a ferrofluid of cobalt ferrite nanoparticles. We show that the embedded magnetic structures significantly increased the magnetic field in the channel, resulting in up to 4-fold enhancement in immunomagnetic capture as compared with a channel without these embedded magnetic structures. We also studied the spatial distribution of trapped cells both experimentally and computationally. We determined that the surface pattern of these trapped cells was determined by both location of the magnet and layout of the in-channel magnetic structures. Our magnetic structure embedded microfluidic device achieved over 90% capture efficiency at a flow velocity of 4 mm/s, a speed that was roughly two orders of magnitude faster than previous microfluidic systems used for a similar purpose. We envision that our technology will provide a powerful tool for detection and enrichment of rare cells from biological samples.

  1. Mapping of Protein-Protein Interactions of E. coli RNA Polymerase with Microfluidic Mechanical Trapping

    PubMed Central

    Bates, Steven R.; Quake, Stephen R.

    2014-01-01

    The biophysical details of how transcription factors and other proteins interact with RNA polymerase are of great interest as they represent the nexus of how structure and function interact to regulate gene expression in the cell. We used an in vitro microfluidic approach to map interactions between a set of ninety proteins, over a third of which are transcription factors, and each of the four subunits of E. coli RNA polymerase, and we compared our results to those of previous large-scale studies. We detected interactions between RNA polymerase and transcription factors that earlier high-throughput screens missed; our results suggest that such interactions can occur without DNA mediation more commonly than previously appreciated. PMID:24643045

  2. MICROFLUIDIC MIXERS FOR THE INVESTIGATION OF PROTEIN FOLDING USING SYNCHROTRON RADIATION CIRCULAR DICHROISM SPECTROSCOPY

    SciTech Connect

    Kane, A; Hertzog, D; Baumgartel, P; Lengefeld, J; Horsley, D; Schuler, B; Bakajin, O

    2006-03-20

    The purpose of this study is to design, fabricate and optimize microfluidic mixers to investigate the kinetics of protein secondary structure formation with Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The mixers are designed to rapidly initiate protein folding reaction through the dilution of denaturant. The devices are fabricated out of fused silica, so that they are transparent in the UV. We present characterization of mixing in the fabricated devices, as well as the initial SRCD data on proteins inside the mixers.

  3. Optically Induced Thermal Gradients for Protein Characterization in Nanolitre-scale Samples in Microfluidic Devices

    PubMed Central

    Sagar, D. M.; Aoudjane, Samir; Gaudet, Matthieu; Aeppli, Gabriel; Dalby, Paul A.

    2013-01-01

    Proteins are the most vital biological functional units in every living cell. Measurement of protein stability is central to understanding their structure, function and role in diseases. While proteins are also sought as therapeutic agents, they can cause diseases by misfolding and aggregation in vivo. Here we demonstrate a novel method to measure protein stability and denaturation kinetics, on unprecedented timescales, through optically-induced heating of nanolitre samples in microfluidic capillaries. We obtain protein denaturation kinetics as a function of temperature, and accurate thermodynamic stability data, from a snapshot experiment on a single sample. We also report the first experimental characterization of optical heating in controlled microcapillary flow, verified by computational fluid dynamics modelling. Our results demonstrate that we now have the engineering science in hand to design integrated all-optical microfluidic chips for a diverse range of applications including in-vitro DNA amplification, healthcare diagnostics, and flow chemistry. PMID:23823279

  4. Fabrication of anti-protein-fouling poly(ethylene glycol) microfluidic chip electrophoresis by sandwich photolithography.

    PubMed

    Cong, Hailin; Xu, Xiaodan; Yu, Bing; Liu, Huwei; Yuan, Hua

    2016-07-01

    Microfluidic chip electrophoresis (MCE) is a powerful separation tool for biomacromolecule analysis. However, adsorption of biomacromolecules, particularly proteins onto microfluidic channels severely degrades the separation performance of MCE. In this paper, an anti-protein-fouling MCE was fabricated using a novel sandwich photolithography of poly(ethylene glycol) (PEG) prepolymers. Photopatterned microchannel with a minimum resolution of 10 μm was achieved. After equipped with a conventional online electrochemical detector, the device enabled baseline separation of bovine serum albumin, lysozyme (Lys), and cytochrome c (Cyt-c) in 53 s under a voltage of 200 V. Compared with a traditional polydimethylsiloxane MCE made by soft lithography, the PEG MCE made by the sandwich photolithography not only eliminated the need of a master mold and the additional modification process of the microchannel but also showed excellent anti-protein-fouling properties for protein separation.

  5. Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

    SciTech Connect

    Emamzadah, Soheila; Petty, Tom J.; De Almeida, Victor; Nishimura, Taisuke; Joly, Jacques; Ferrer, Jean-Luc; Halazonetis, Thanos D.

    2009-09-01

    A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction. Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus’ molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.

  6. Dynamics of Drosophila embryonic patterning network perturbed in space and time using microfluidics

    NASA Astrophysics Data System (ADS)

    Lucchetta, Elena M.; Lee, Ji Hwan; Fu, Lydia A.; Patel, Nipam H.; Ismagilov, Rustem F.

    2005-04-01

    Biochemical networks are perturbed both by fluctuations in environmental conditions and genetic variation. These perturbations must be compensated for, especially when they occur during embryonic pattern formation. Complex chemical reaction networks displaying spatiotemporal dynamics have been controlled and understood by perturbing their environment in space and time. Here, we apply this approach using microfluidics to investigate the robust network in Drosophila melanogaster that compensates for variation in the Bicoid morphogen gradient. We show that the compensation system can counteract the effects of extremely unnatural environmental conditions-a temperature step-in which the anterior and posterior halves of the embryo are developing at different temperatures and thus at different rates. Embryonic patterning was normal under this condition, suggesting that a simple reciprocal gradient system is not the mechanism of compensation. Time-specific reversals of the temperature step narrowed down the critical period for compensation to between 65 and 100min after onset of embryonic development. The microfluidic technology used here may prove useful to future studies, as it allows spatial and temporal regulation of embryonic development.

  7. Electrokinetic protein preconcentration using a simple glass/poly(dimethylsiloxane) microfluidic chip.

    PubMed

    Kim, Sun Min; Burns, Mark A; Hasselbrink, Ernest F

    2006-07-15

    We discovered that a protein concentration device can be constructed using a simple one-layer fabrication process. Microfluidic half-channels are molded using standard procedures in PDMS; the PDMS layer is reversibly bonded to a glass base such as a microscope slide. The microfluidic channels are chevron-shaped, in mirror image orientation, with their apexes designed to pass within approximately 20 microm of each other, forming a thin-walled section between the channels. When an electric field is applied across this thin-walled section, negatively charged proteins are observed to concentrate on the anode side of it. About 10(3)-10(6)-fold protein concentration was achieved in 30 min. Subsequent separation of two different concentrated proteins is easily achieved by switching the direction of the electric field in the direction parallel to the thin-walled section. We hypothesize that a nanoscale channel forms between the PDMS and the glass due to the weak, reversible bonding method. This hypothesis is supported by the observation that, when the PDMS and glass are irreversibly bonded, this phenomenon is not observed until a very high E-field was applied and dielectric breakdown of the PDMS is observed. We therefore suspect that the ion exclusion-enrichment effect caused by electrical double layer overlapping induces cationic selectivity of this nanochannel. This simple on-chip protein preconcentration and separation device could be a useful component in practically any PDMS-on-glass microfluidic device used for protein assays.

  8. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    PubMed Central

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Berger, James M.

    2015-01-01

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources. PMID:25849403

  9. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array.

    PubMed

    Lyubimov, Artem Y; Murray, Thomas D; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B; Cohen, Aina E; Soltis, S Michael; Baxter, Elizabeth L; Brewster, Aaron S; Sauter, Nicholas K; Brunger, Axel T; Berger, James M

    2015-04-01

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  10. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    SciTech Connect

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; Sauter, Nicholas K.; Brunger, Axel T.; Berger, James M.

    2015-03-27

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat for conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.

  11. Capture and X-ray diffraction studies of protein microcrystals in a microfluidic trap array

    DOE PAGES

    Lyubimov, Artem Y.; Murray, Thomas D.; Koehl, Antoine; Araci, Ismail Emre; Uervirojnangkoorn, Monarin; Zeldin, Oliver B.; Cohen, Aina E.; Soltis, S. Michael; Baxter, Elizabeth L.; Brewster, Aaron S.; et al

    2015-03-27

    X-ray free-electron lasers (XFELs) promise to enable the collection of interpretable diffraction data from samples that are refractory to data collection at synchrotron sources. At present, however, more efficient sample-delivery methods that minimize the consumption of microcrystalline material are needed to allow the application of XFEL sources to a wide range of challenging structural targets of biological importance. Here, a microfluidic chip is presented in which microcrystals can be captured at fixed, addressable points in a trap array from a small volume (<10 µl) of a pre-existing slurry grown off-chip. The device can be mounted on a standard goniostat formore » conducting diffraction experiments at room temperature without the need for flash-cooling. Proof-of-principle tests with a model system (hen egg-white lysozyme) demonstrated the high efficiency of the microfluidic approach for crystal harvesting, permitting the collection of sufficient data from only 265 single-crystal still images to permit determination and refinement of the structure of the protein. This work shows that microfluidic capture devices can be readily used to facilitate data collection from protein microcrystals grown in traditional laboratory formats, enabling analysis when cryopreservation is problematic or when only small numbers of crystals are available. Such microfluidic capture devices may also be useful for data collection at synchrotron sources.« less

  12. A microfluidic linear node array for the study of protein-ligand interactions.

    PubMed

    Li, Cheuk-Wing; Yu, Guodong; Jiang, Jingyun; Lee, Simon Ming-Yuen; Yi, Changqing; Yue, Wanqing; Yang, Mengsu

    2014-10-21

    We have developed a microfluidic device for the continuous separation of small molecules from a protein mixture and demonstrated its practical use in the study of protein-ligand binding, a crucial aspect in drug discovery. Our results demonstrated dose-dependent binding between bovine serum albumin (BSA) and its small-molecule site marker, Eosin Y (EY), and found that the binding reached a plateau when the BSA : EY ratio was above 1, which agreed with the eosin binding capacity of BSA reported in literature. By streamline control using a combination of two fundamental building blocks (R and L nodes) with a microdevice operated at a high flow rate (up to 1300 μL h(-1)), a solution barrier was created to "filter" off protein/protein-ligand complexes such that the small unbound molecules were isolated and quantified easily. The percentage decrease of small molecules with increasing protein concentration indicated the presence of binding events. Several fluorophores with different molecular weights were used to test the performance of the microfluidic "filter", which was tunable by 1) the total flow rate, and/or 2) the flow distribution ratio between the two device inlets; both were easily controllable by changing the syringe pump settings. Since the microdevice was operated at a relatively high flow rate, aliquots were easily recovered from the device outlets to facilitate off-chip detection. This microfluidic design is a novel and promising tool for preliminary drug screening.

  13. Fluorescence enhancement and multiple protein detection in ZnO nanostructure microfluidic devices.

    PubMed

    Sang, Chen-Hsiang; Chou, Shu-Jen; Pan, F M; Sheu, Jeng-Tzong

    2016-01-15

    In this study, different morphological ZnO nanostructures, those of sharp nanowires (NWs), rod NWs, and hexahedral-puncheon nanostructures, were grown in microfluidic channels on the same glass substrate. Characterizations of correspondent biomolecule binding properties were simulated and demonstrated. The surface was modified using 3-ammineopropyl-triethoxysilane (3-APTES) and biotin-N-hydroxysuccinimide ester (NHS-biotin). Different concentrations (4.17pM to 41.7nM) of dye-conjugated streptavidin were simultaneously infused through the second microfluidic channels, which lie 90° from the first microfluidic channels. The florescent intensity at the crossover areas showed good agreement with simulations, with sharp ZnO NWs exhibiting the largest dynamic range and the highest fluorescent intensity. We further characterize correspondent protein detection using sharp ZnO NWs. The surfaces of these ZnO NWs were modified with mouse immunoglobulin G (IgG), infused through the second microfluidic channels with dye-conjugated (Alexa 546) anti-mouse IgG in different concentrations. Concentrations ranging from 417fM to 41.7nM can be resolved using sharp ZnO NWs. Finally, multiple protein detection was demonstrated using a five-by-eight microfluidic channel array. Fluorescence images present clear multiple detections at the crossover areas when using the sharp ZnO NWs for simultaneous dye-conjugated anti-mouse IgG and dye-conjugated anti-rabbit IgG (Alexa 647) detection. PMID:26322591

  14. Recombinant Protein-Stabilized Monodisperse Microbubbles with Tunable Size Using a Valve-Based Microfluidic Device

    PubMed Central

    2015-01-01

    Microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. Conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. Stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. Here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. Our microfluidic device uses an air-actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. The size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. The microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. We show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. Our protein-shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound-related applications. PMID:25265041

  15. Microfluidic chips with multi-junctions: an advanced tool in recovering proteins from inclusion bodies.

    PubMed

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2015-01-01

    Active recombinant proteins are used for studying the biological functions of genes and for the development of therapeutic drugs. Overexpression of recombinant proteins in bacteria often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. Protein refolding is an important process for obtaining active recombinant proteins from inclusion bodies. However, the conventional refolding method of dialysis or dilution is time-consuming and recovered active protein yields are often low, and a cumbersome trial-and-error process is required to achieve success. To circumvent these difficulties, we used controllable diffusion through laminar flow in microchannels to regulate the denaturant concentration. This method largely aims at reducing protein aggregation during the refolding procedure. This Commentary introduces the principles of the protein refolding method using microfluidic chips and the advantage of our results as a tool for rapid and efficient recovery of active recombinant proteins from inclusion bodies.

  16. Maximizing Fibroblast Adhesion on Protein-Coated Surfaces Using Microfluidic Cell Printing

    PubMed Central

    Davidoff, S.N.; Au, D.; Gale, B.K.; Brooks, B.D.; Brooks, A.E.

    2015-01-01

    translation of in vitro cell based assays to in vivo cellular response is imprecise at best. The advent of three-dimensional cell cultures in addition to bioreactor type microfluidics has improved the situation. However, these technical advances cannot be easily combined due to practical limitations. Development of a vertical microfluidic cell printer overcomes this obstacle, providing the ability to more closely recapitulate complex cellular environments and responses. As a proof of concept, we investigated the adhesion of fibroblasts under flow on protein-coated surfaces using a novel vertical microfluidic print head to isolate and manipulate both mechanical and biological factors as a model of fibroblast behavior during the foreign body response following implant insertion. A low flow rate with larger microfluidic channels onto a serum-coated surface has been determined to allow the highest density of viable fibroblasts to attach to the surface. While these insights into fibroblast surface attachment may lead to better material designs, the methods developed herein will certainly be useful as a biomaterials testing platform. PMID:26989480

  17. Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery.

    PubMed

    Pessi, Jenni; Santos, Hélder A; Miroshnyk, Inna; JoukoYliruusi; Weitz, David A; Mirza, Sabiruddin

    2014-09-10

    In this study, microfluidic technology was employed to develop protein formulations. The microcapsules were produced with a biphasic flow to create water-oil-water (W/O/W) double emulsion droplets with ultrathin shells. Optimized microcapsule formulations containing 1% (w/w) bovine serum albumin (BSA) in the inner phase were prepared with poly(vinyl alcohol), polycaprolactone and polyethylene glycol. All the particles were found to be intact and with a particle size of 23-47 μm. Furthermore, the particles were monodisperse, non-porous and stable up to 4 weeks. The encapsulation efficiency of BSA in the microcapsules was 84%. The microcapsules released 30% of their content within 168 h. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins.

  18. Photopolymerized sol-gel monoliths for separations of glycosylated proteins and peptides in microfluidic chips.

    PubMed

    Levy, Miriam H; Plawsky, Joel; Cramer, Steven M

    2013-07-01

    Photopolymerized silica sol-gel monoliths, functionalized with boronic acid ligands, have been developed for protein and peptide separations in polydimethylsiloxane microfluidic devices. Pore size characterization of the monoliths was carried out with SEM, image analysis, and differential scanning calorimetry to evaluate both the micron-sized macropores and the nanometer-sized mesopores. Monoliths were functionalized with boronic acid using three different immobilization techniques. Batch experiments were conducted to determine the capacity of the monoliths and selectivity toward cis-diol-containing compounds. Conalbumin was used as a model glycoprotein, and a tryptic digest of the glycoprotein horseradish peroxidase was used as a peptide mixture to demonstrate proof-of-concept extraction of glycoproteins and glycopeptides by the monoliths formulated in polydimethylsiloxane microfluidic chips. For proteins, fluorescence detection was used, whereas the peptide separations employed off-line analysis using MALDI-MS. PMID:23703808

  19. Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability.

    PubMed

    Davis, Lloyd M; Lubbeck, Jennifer L; Dean, Kevin M; Palmer, Amy E; Jimenez, Ralph

    2013-06-21

    This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries. PMID:23636097

  20. Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability

    PubMed Central

    Davis, Lloyd M.; Lubbeck, Jennifer L.; Dean, Kevin M.; Palmer, Amy E.; Jimenez, Ralph

    2014-01-01

    This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries. PMID:23636097

  1. Crystallization of the large membrane protein complex photosystem I in a microfluidic channel.

    PubMed

    Abdallah, Bahige G; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2013-12-23

    Traditional macroscale protein crystallization is accomplished nontrivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time-consuming and resource-intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these "difficult to crystallize" proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via second-order nonlinear imaging of chiral crystals (SONICC) was applied for the detection of nanometer- and micrometer-sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.

  2. Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel

    PubMed Central

    Abdallah, Bahige G.; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

    2014-01-01

    Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ‘difficult to crystallize’ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. PMID:24191698

  3. Nanoliter-scale protein crystallization and screening with a microfluidic droplet robot.

    PubMed

    Zhu, Ying; Zhu, Li-Na; Guo, Rui; Cui, Heng-Jun; Ye, Sheng; Fang, Qun

    2014-05-23

    Large-scale screening of hundreds or even thousands of crystallization conditions while with low sample consumption is in urgent need, in current structural biology research. Here we describe a fully-automated droplet robot for nanoliter-scale crystallization screening that combines the advantages of both automated robotics technique for protein crystallization screening and the droplet-based microfluidic technique. A semi-contact dispensing method was developed to achieve flexible, programmable and reliable liquid-handling operations for nanoliter-scale protein crystallization experiments. We applied the droplet robot in large-scale screening of crystallization conditions of five soluble proteins and one membrane protein with 35-96 different crystallization conditions, study of volume effects on protein crystallization, and determination of phase diagrams of two proteins. The volume for each droplet reactor is only ca. 4-8 nL. The protein consumption significantly reduces 50-500 fold compared with current crystallization stations.

  4. Microfluidic crystallization.

    PubMed

    Leng, Jacques; Salmon, Jean-Baptiste

    2009-01-01

    Microfluidics offers a wide range of new tools that permit one to revisit the formation of crystals in solution and yield insights into crystallization processes. We review such recent microfluidic devices and particularly emphasize lab-on-chips dedicated to the high-throughput screening of crystallization conditions of proteins with nanolitre consumption. We also thoroughly discuss the possibilities offered by the microfluidic tools to acquire thermodynamic and kinetic data that may improve industrial processes and shed a new light on nucleation and growth mechanisms.

  5. Online multi-channel microfluidic chip-mass spectrometry and its application for quantifying noncovalent protein-protein interactions.

    PubMed

    Liu, Wu; Chen, Qiushui; Lin, Xuexia; Lin, Jin-Ming

    2015-03-01

    To establish an automatic and online microfluidic chip-mass spectrometry (chip-MS) system, a device was designed and fabricated for microsampling by a hybrid capillary. The movement of the capillary was programmed by a computer to aspirate samples from different microfluidic channels in the form of microdroplets (typically tens of nanoliters in volume), which were separated by air plugs. The droplets were then directly analyzed by MS via paper spray ionization without any pretreatment. The feasibility and performance were demonstrated by a concentration gradient experiment. Furthermore, after eliminating the effect of nonuniform response factors by an internal standard method, determination of the association constant within a noncovalent protein-protein complex was successfully accomplished with the MS-based titration indicating the versatility and the potential of this novel platform for widespread applications. PMID:25597452

  6. Rapid detection and quantification of specific proteins by immunodepletion and microfluidic separation

    PubMed Central

    Azadi, Glareh; Gustafson, Eric; Wessel, Gary M.; Tripathi, Anubhav

    2012-01-01

    Conventional immunoblotting techniques are labor intensive, time consuming and rely on the elution of target protein after depletion. Here we describe a new method for detection and quantification of proteins, independent of washing and elution. In this method, the target protein is first captured by immunodepletion with antibody coated microbeads. In the second step, both the supernatant after immunodepletion and the untreated protein sample are directly analyzed by microfluidic electrophoresis without further processing. Subsequently, the detection and quantification are performed comparing the electropherograms of these two samples. This method was tested using an Escherichia coli lysate with a FLAG-tagged protein and anti-FLAG magnetic beads. An incubation of as little as one minute was sufficient for detectable depletion (66%) by microchip electrophoresis. Longer incubation (up to 60 minutes) resulted in more depletion of the target band (82%). Our results show that only 19% of the target is recovered after elution from the beads. By eliminating multiple wash and elution steps, our method is faster, less labor intensive, and highly reproducible. Even in case of non-specific binding at low concentrations, the target protein can be easily identified. This work highlights the advantages of integrating immunodepletion techniques on a microfluidic platform. PMID:22539461

  7. Rapid fabrication of microfluidic polymer electrolyte membrane fuel cell in PDMS by surface patterning of perfluorinated ion-exchange resin

    NASA Astrophysics Data System (ADS)

    Song, Yong-Ak; Batista, Candy; Sarpeshkar, Rahul; Han, Jongyoon

    In this paper we demonstrate a simple and rapid fabrication method for a microfluidic polymer electrolyte membrane (PEM) fuel cell using polydimethylsiloxane (PDMS), which has become the de facto standard material in BioMEMS. Instead of integrating a Nafion sheet film between two layers of a PDMS device in a traditional "sandwich format," we pattern a perfluorinated ion-exchange resin such as a Nafion resin on a glass substrate using a reversibly bonded PDMS microchannel to generate an ion-selective membrane between the fuel-cell electrodes. After this patterning step, the assembly of the microfluidic fuel cell is accomplished by simple oxygen plasma bonding between the PDMS chip and the glass substrate. In an example implementation, the planar PEM microfluidic fuel cell generates an open circuit voltage of 600-800 mV and delivers a maximum current output of nearly 4 μA. To enhance the power output of the fuel cell we utilize self-assembled colloidal arrays as a support matrix for the Nafion resin. Such arrays allow us to increase the thickness of the ion-selective membrane to 20 μm and increase the current output by 166%. Our novel fabrication method enables rapid prototyping of microfluidic fuel cells to study various ion-exchange resins for the polymer electrolyte membrane. Our work will facilitate the development of miniature, implantable, on-chip power sources for biomedical applications.

  8. Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics

    PubMed Central

    Sarkar, Aniruddh; Hou, Han Wei; Mahan, Alison. E.; Han, Jongyoon; Alter, Galit

    2016-01-01

    Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary ‘bind-elute’ separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets–cells or proteins–bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients. PMID:27026280

  9. Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast

    PubMed Central

    Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L.; Hallström, Björn M.; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N.; Andersson-Svahn, Helene; Nielsen, Jens

    2015-01-01

    There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories. PMID:26261321

  10. Microfluidic devices for label-free separation of cells through transient interaction with asymmetric receptor patterns

    NASA Astrophysics Data System (ADS)

    Bose, S.; Singh, R.; Hollatz, M. H.; Lee, C.-H.; Karp, J.; Karnik, R.

    2012-02-01

    Cell sorting serves an important role in clinical diagnosis and biological research. Most of the existing microscale sorting techniques are either non-specific to antigen type or rely on capturing cells making sample recovery difficult. We demonstrate a simple; yet effective technique for isolating cells in an antigen specific manner by using transient interactions of the cell surface antigens with asymmetric receptor patterned surface. Using microfluidic devices incorporating P-selectin patterns we demonstrate separation of HL60 cells from K562 cells. We achieved a sorting purity above 90% and efficiency greater than 85% with this system. We also present a mathematical model incorporating flow mediated and adhesion mediated transport of cells in the microchannel that can be used to predict the performance of these devices. Lastly, we demonstrate the clinical significance of the method by demonstrating single step separation of neutrophils from whole blood. When whole blood is introduced in the device, the granulocyte population gets separated exclusively yielding neutrophils of high purity (<10% RBC contamination). To our knowledge, this is the first ever demonstration of continuous label free sorting of neutrophils from whole blood. We believe this technology will be useful in developing point-of-care diagnostic devices and also for a host of cell sorting applications.

  11. Microfluidics-Based Selection of Red-Fluorescent Proteins with Decreased Rates of Photobleaching

    PubMed Central

    Dean, Kevin M.; Lubbeck, Jennifer L.; Davis, Lloyd M.; Regmi, Chola K.; Chapagain, Prem P.; Gerstman, Bernard S.; Jimenez, Ralph; Palmer, Amy E.

    2014-01-01

    Fluorescent proteins offer exceptional labeling specificity in living cells and organisms. Unfortunately, their photophysical properties remain far from ideal for long-term imaging of low-abundance cellular constituents, in large part because of their poor photostability. Despite widespread engineering efforts, improving the photostability of fluorescent proteins remains challenging due to lack of appropriate high-throughput selection methods. Here, we use molecular dynamics guided mutagenesis in conjunction with a recently developed microfluidic-based platform, which sorts cells based on their fluorescence photostability, to identify red fluorescent proteins with decreased photobleaching from a HeLa cell-based library. The identified mutant, named Kriek, has 2.5- and 4-fold higher photostability than its progenitor, mCherry, under widefield and confocal illumination, respectively. Furthermore, the results provide insight into mechanisms for enhancing photostability and their connections with other photophysical processes, thereby providing direction for ongoing development of fluorescent proteins with improved single-molecule and low-copy imaging capabilities. Insight, innovation, integration Fluorescent proteins enable imaging in situ, throughout the visible spectrum, with superb molecular specificity and single-molecule sensitivity. Unfortunately, when compared to leading small-molecule fluorophores (e.g., Cy3), fluorescent proteins, suffer from accelerated photobleaching and poor integrated photon output. This results from a lack of appropriate high-throughput methods for improving the photostability of fluorescent proteins, as well as a poor molecular understanding of fluorescent protein photobleaching. Here, we report the first application of a recently developed microfluidic cell-sorter to identify fluorescent proteins from a mCherry-derived library with improved photostability. The results provide insight into fluorescent protein photophysics, greatly

  12. Paper-based microfluidic approach for surface-enhanced raman spectroscopy and highly reproducible detection of proteins beyond picomolar concentration.

    PubMed

    Saha, Arindam; Jana, Nikhil R

    2015-01-14

    Although microfluidic approach is widely used in various point of care diagnostics, its implementation in surface enhanced Raman spectroscopy (SERS)-based detection is challenging. This is because SERS signal depends on plasmonic nanoparticle aggregation induced generation of stable electromagnetic hot spots and in currently available microfluidic platform this condition is difficult to adapt. Here we show that SERS can be adapted using simple paper based microfluidic system where both the plasmonic nanomaterials and analyte are used in mobile phase. This approach allows analyte induced controlled particle aggregation and electromagnetic hot spot generation inside the microfluidic channel with the resultant SERS signal, which is highly reproducible and sensitive. This approach has been used for reproducible detection of protein in the pico to femtomolar concentration. Presented approach is simple, rapid, and cost-effective, and requires low sample volume. Method can be extended for SERS-based detection of other biomolecules.

  13. Electrochemical Protein Cleavage in a Microfluidic Cell with Integrated Boron Doped Diamond Electrodes.

    PubMed

    van den Brink, Floris T G; Zhang, Tao; Ma, Liwei; Bomer, Johan; Odijk, Mathieu; Olthuis, Wouter; Permentier, Hjalmar P; Bischoff, Rainer; van den Berg, Albert

    2016-09-20

    Specific electrochemical cleavage of peptide bonds at the C-terminal side of tyrosine and tryptophan generates peptides amenable to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification. To this end we developed a microfluidic electrochemical cell of 160 nL volume that combines a cell geometry optimized for a high electrochemical conversion efficiency (>95%) with an integrated boron doped diamond (BDD) working electrode offering a wide potential window in aqueous solution and reduced adsorption of peptides and proteins. Efficient cleavage of the proteins bovine insulin and chicken egg white lysozyme was observed at 4 out of 4 and 7 out of 9 of the predicted cleavage sites, respectively. Chicken egg white lysozyme was identified based on 5 electrochemically generated peptides using a proteomics database searching algorithm. These results show that electrochemical peptide bond cleavage in a microfluidic cell is a novel, fully instrumental approach toward protein analysis and eventually proteomics studies in conjunction with mass spectrometry. PMID:27563730

  14. Science Issues Associated with the Use of a Microfluidic Chip Designed Specifically for Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Holmes, Anna M.; Monaco, Lisa; Barnes, Cindy; Spearing, Scott; Jenkins, Andy; Johnson, Todd; Mayer, Derek; Cole, Helen

    2003-01-01

    The Iterative Biological Crystallization team in partnership with Caliper Technologies has produced a prototype microfluidic chip for batch crystallization that has been designed and tested. The chip is designed for the mixing and dispensing of up to five solutions with possible variation of the recipe being delivered to two growth wells. Developments that have led to the successful on-chip crystallization of a few model proteins have required investigative insight into many different areas, including fluid mixing dynamics, surface treatments, quantification and fidelity of reagent delivery. This presentation will encompass the ongoing studies and data accumulated toward these efforts.

  15. Re-use of commercial microfluidics chips for DNA, RNA, and protein electrophoresis.

    PubMed

    Nguyen, Thi; Kwak, Sukyoung; Karpowicz, Steven J

    2014-11-01

    Microfluidics chip technology is a powerful and convenient alternative to agarose gels and PAGE, but costs can be high due to certain chips being non-reusable. Here we describe a method to regenerate, re-use, and store Agilent DNA, RNA, and protein electrophoresis chips designed for use in the Bioanalyzer 2100. By washing the sample wells and displacing the old gel matrix with new gel-dye mix, we have run samples on the same chip up to ten times with negligible loss of signal quality. Chips whose wells were loaded with buffer or water were stored successfully for one week before re-use.

  16. Patterned electrode-based amperometric gas sensor for direct nitric oxide detection within microfluidic devices.

    PubMed

    Cha, Wansik; Tung, Yi-Chung; Meyerhoff, Mark E; Takayama, Shuichi

    2010-04-15

    This article describes a thin amperometric nitric oxide (NO) sensor that can be microchannel embedded to enable direct real-time detection of NO produced by cells cultured within the microdevice. A key for achieving the thin ( approximately 1 mm) planar sensor configuration required for sensor-channel integration is the use of gold/indium-tin oxide patterned electrode directly on a porous polymer membrane (pAu/ITO) as the base working electrode. The electrochemically deposited Au-hexacyanoferrate layer on pAu/ITO is used to catalyze NO oxidation to nitrite at lower applied potentials (0.65-0.75 V vs Ag/AgCl) and stabilize current output. Furthermore, use of a gas-permeable membrane to separate internal sensor compartments from the sample phase imparts excellent NO selectivity over common interfering agents (e.g., nitrite, ascorbate, ammonia, etc.) present in culture media and biological fluids. The optimized sensor design reversibly detects NO down to the approximately 1 nM level in stirred buffer and <10 nM in flowing buffer when integrated within a polymeric microfluidic device. We demonstrate utility of the channel-embedded sensor by monitoring NO generation from macrophages cultured within non-gas-permeable microchannels, as they are stimulated with endotoxin. PMID:20329749

  17. Development-on-chip: in vitro neural tube patterning with a microfluidic device

    PubMed Central

    Soundararajan, Prabakaran; Chennampally, Phaneendra; Cox, Gregory A.

    2016-01-01

    Embryogenesis is a highly regulated process in which the precise spatial and temporal release of soluble cues directs differentiation of multipotent stem cells into discrete populations of specialized adult cell types. In the spinal cord, neural progenitor cells are directed to differentiate into adult neurons through the action of mediators released from nearby organizing centers, such as the floor plate and paraxial mesoderm. These signals combine to create spatiotemporal diffusional landscapes that precisely regulate the development of the central nervous system (CNS). Currently, in vivo and ex vivo studies of these signaling factors present some inherent ambiguity. In vitro methods are preferred for their enhanced experimental clarity but often lack the technical sophistication required for biological realism. In this article, we present a versatile microfluidic platform capable of mimicking the spatial and temporal chemical environments found in vivo during neural tube development. Simultaneous opposing and/or orthogonal gradients of developmental morphogens can be maintained, resulting in neural tube patterning analogous to that observed in vivo. PMID:27246712

  18. Inkjet patterned superhydrophobic paper for open-air surface microfluidic devices.

    PubMed

    Elsharkawy, Mohamed; Schutzius, Thomas M; Megaridis, Constantine M

    2014-03-21

    We present a facile approach for the fabrication of low-cost surface biomicrofluidic devices on superhydrophobic paper created by drop-casting a fluoroacrylic copolymer onto microtextured paper. Wettability patterning is performed with a common household printer, which produces regions of varying wettability by simply controlling the intensity of ink deposited over prespecified domains. The procedure produces surfaces that are capable of selective droplet sliding and adhesion, when inclined. Using this methodology, we demonstrate the ability to tune the sliding angles of 10 μL water droplets in the range from 13° to 40° by printing lines of constant ink intensity and varied width from 0.1 mm to 2 mm. We also formulate a simple model to predict the onset of droplet sliding on printed lines of known width and wettability. Experiments demonstrate open-air surface microfluidic devices that are capable of pumpless transport, mixing and rapid droplet sampling (~0.6 μL at 50 Hz). Lastly, post treatment of printed areas with pH indicator solutions exemplifies the utility of these substrates in point-of-care diagnostics, which are needed at geographical locations where access to sophisticated testing equipment is limited or non-existent.

  19. Mass spectrometry-based monitoring of millisecond protein-ligand binding dynamics using an automated microfluidic platform.

    PubMed

    Cong, Yongzheng; Katipamula, Shanta; Trader, Cameron D; Orton, Daniel J; Geng, Tao; Baker, Erin S; Kelly, Ryan T

    2016-04-26

    Characterizing protein-ligand binding dynamics is crucial for understanding protein function and for developing new therapeutic agents. We present a novel microfluidic platform that features rapid mixing of protein and ligand solutions, variable incubation times, and an integrated electrospray ionization source for mass spectrometry-based monitoring of protein-ligand binding dynamics. This platform offers many advantages, including solution-based binding, label-free detection, automated operation, rapid mixing, and low sample consumption. PMID:27009517

  20. Growth of Diffraction-Quality Protein Crystals Using a Harvestable Microfluidic Device

    PubMed Central

    2015-01-01

    Protein crystallization is the major bottleneck in the entire process of protein crystallography, and obtaining diffraction-quality crystals can be unpredictable and sometimes exceptionally difficult, requiring many rounds of high-throughput screening. Recently, a more time- and cost-saving strategy to use the commercially available microfluidic devices called Crystal Formers has emerged. Herein we show the application of such a device using a protein from Legionella pneumophila called LidL that is predicted to be involved in the ability to efficiently manipulate host cell trafficking events once internalized by the host cell. After setting up just one 96-channel Crystal Former tray, we were able to obtain a diffraction-quality crystal that diffracted to 2.76 Å. These results show that Crystal Formers can be used to screen and optimize crystals to directly produce crystals for structure determination. PMID:25013386

  1. Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth

    PubMed Central

    Celik, Yeliz; Drori, Ran; Pertaya-Braun, Natalya; Altan, Aysun; Barton, Tyler; Bar-Dolev, Maya; Groisman, Alex; Davies, Peter L.; Braslavsky, Ido

    2013-01-01

    Antifreeze proteins (AFPs) are a subset of ice-binding proteins that control ice crystal growth. They have potential for the cryopreservation of cells, tissues, and organs, as well as for production and storage of food and protection of crops from frost. However, the detailed mechanism of action of AFPs is still unclear. Specifically, there is controversy regarding reversibility of binding of AFPs to crystal surfaces. The experimentally observed dependence of activity of AFPs on their concentration in solution appears to indicate that the binding is reversible. Here, by a series of experiments in temperature-controlled microfluidic devices, where the medium surrounding ice crystals can be exchanged, we show that the binding of hyperactive Tenebrio molitor AFP to ice crystals is practically irreversible and that surface-bound AFPs are sufficient to inhibit ice crystal growth even in solutions depleted of AFPs. These findings rule out theories of AFP activity relying on the presence of unbound protein molecules. PMID:23300286

  2. Microfluidic Cold-Finger Device for the Investigation of Ice-Binding Proteins.

    PubMed

    Haleva, Lotem; Celik, Yeliz; Bar-Dolev, Maya; Pertaya-Braun, Natalya; Kaner, Avigail; Davies, Peter L; Braslavsky, Ido

    2016-09-20

    Ice-binding proteins (IBPs) bind to ice crystals and control their structure, enlargement, and melting, thereby helping their host organisms to avoid injuries associated with ice growth. IBPs are useful in applications where ice growth control is necessary, such as cryopreservation, food storage, and anti-icing. The study of an IBP's mechanism of action is limited by the technological difficulties of in situ observations of molecules at the dynamic interface between ice and water. We describe herein a new, to our knowledge, apparatus designed to generate a controlled temperature gradient in a microfluidic chip, called a microfluidic cold finger (MCF). This device allows growth of a stable ice crystal that can be easily manipulated with or without IBPs in solution. Using the MCF, we show that the fluorescence signal of IBPs conjugated to green fluorescent protein is reduced upon freezing and recovers at melting. This finding strengthens the evidence for irreversible binding of IBPs to their ligand, ice. We also used the MCF to demonstrate the basal-plane affinity of several IBPs, including a recently described IBP from Rhagium inquisitor. Use of the MCF device, along with a temperature-controlled setup, provides a relatively simple and robust technique that can be widely used for further analysis of materials at the ice/water interface.

  3. Microfluidic Cold-Finger Device for the Investigation of Ice-Binding Proteins.

    PubMed

    Haleva, Lotem; Celik, Yeliz; Bar-Dolev, Maya; Pertaya-Braun, Natalya; Kaner, Avigail; Davies, Peter L; Braslavsky, Ido

    2016-09-20

    Ice-binding proteins (IBPs) bind to ice crystals and control their structure, enlargement, and melting, thereby helping their host organisms to avoid injuries associated with ice growth. IBPs are useful in applications where ice growth control is necessary, such as cryopreservation, food storage, and anti-icing. The study of an IBP's mechanism of action is limited by the technological difficulties of in situ observations of molecules at the dynamic interface between ice and water. We describe herein a new, to our knowledge, apparatus designed to generate a controlled temperature gradient in a microfluidic chip, called a microfluidic cold finger (MCF). This device allows growth of a stable ice crystal that can be easily manipulated with or without IBPs in solution. Using the MCF, we show that the fluorescence signal of IBPs conjugated to green fluorescent protein is reduced upon freezing and recovers at melting. This finding strengthens the evidence for irreversible binding of IBPs to their ligand, ice. We also used the MCF to demonstrate the basal-plane affinity of several IBPs, including a recently described IBP from Rhagium inquisitor. Use of the MCF device, along with a temperature-controlled setup, provides a relatively simple and robust technique that can be widely used for further analysis of materials at the ice/water interface. PMID:27653473

  4. Microfluidic Electrochemical Immunoarray for Ultrasensitive Detection of Two Cancer Biomarker Proteins in Serum

    PubMed Central

    Chikkaveeraiah, Bhaskara V.; Mani, Vigneshwaran; Patel, Vyomesh; Gutkind, J. Silvio; Rusling, James F.

    2011-01-01

    A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL−1 levels. MPs were conjugated with ~90,000 antibodies and ~200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL−1 for PSA and 0.30 pg mL−1 for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily-labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers. PMID:21632234

  5. Fabrication of a microfluidic device for simultaneous patterning of multiple chemical species by Dip Pen Nanolithography (DPN)

    NASA Astrophysics Data System (ADS)

    Rivas-Cordona, J. Alberto; Banerjee, Debjyoti

    2006-05-01

    This paper describes the fabrication and testing of a microfluidic ink delivery device for Dip Pen Nanolithography (DPN TM). The purpose of this microfluidic device is to maximize the number of chemical species (inks) for nanofabrication that can be simultaneously patterned by DPN. The device (called 'Centiwells') consists of a two-dimensional array of 96micro-wells micro-machined on a silicon substrate and a thermoelectric module attached to the bottom of the substrate. Microbeads of a hygroscopic material (Poly-Ethylene Glycol) are dispensed into the microwells. By reducing the temperature of the substrate to below the dew point water droplets are condensed on the PEG microbeads, dissolving the beads and creating PEG solutions. Following the formation of the PEG solutions, an AFM tip (pen) is lowered into the micro-wells for loading ink ('dipping' or 'inking' step) and subsequently nanolithography is performed.

  6. Lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis.

    PubMed

    Funakoshi, Kei; Suzuki, Hiroaki; Takeuchi, Shoji

    2006-12-15

    Artificial planar lipid bilayers are a powerful tool for the functional study of membrane proteins, yet they have not been widely used due to their low stability and reproducibility. This paper describes an accessible method to form a planar lipid bilayer, simply by contacting two monolayers assembled at the interface between water and organic solvent in a microfluidic chip. The membrane of an organic solvent containing phospholipids at the interface was confirmed to be a bilayer by the capacitance measurement and by measuring the ion channel signal from reconstituted antibiotic peptides. We present two different designs for bilayer formation. One equips two circular wells connected, in which the water/solvent/water interface was formed by simply injecting a water droplet into each well. Another equips the cross-shaped microfluidic channel. In the latter design, formation of the interface at the sectional area was controlled by external syringe pumps. Both methods are extremely simple and reproducible, especially in microdevices, and will lead to automation and multiple bilayer formation for the high-throughput screening of membrane transport in physiological and pharmaceutical studies.

  7. Protein immobilization on the surface of polydimethylsiloxane and polymethyl methacrylate microfluidic devices.

    PubMed

    Khnouf, Ruba; Karasneh, Dina; Albiss, Borhan Aldeen

    2016-02-01

    PDMS and PMMA are two of the most used polymers in the fabrication of lab-on-chip or microfluidic devices. In order to use these polymers in biological applications, it is sometimes essential to be able to bind biomolecules such as proteins and DNA to the surface of these materials. In this work, we have evaluated a number of processes that have been developed to bind protein to PDMS surfaces which include passive adsorption, passive adsorption with glutaraldehyde cross-linking, (3-aminopropyl) triethoxysilane functionalization followed by glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride cross-linkers. It has been shown that the latter technique--using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride--results in more than twice the bonding of protein to the surface of PDMS microchannels than proteins binding passively. We have also evaluated a few techniques that have been tested for the functionalization of PMMA microchannels where we have found that the use of polyethyleneimine (PEI) has led to the strongest protein-PMMA microchannel bond. We finally demonstrated the effect of PDMS curing methodology on protein adsorption to its surface, and showed that increased curing time is the factor that reduces passive adsorption the most.

  8. A method to integrate patterned electrospun fibers with microfluidic systems to generate complex microenvironments for cell culture applications

    PubMed Central

    Wallin, Patric; Zandén, Carl; Carlberg, Björn; Hellström Erkenstam, Nina; Liu, Johan; Gold, Julie

    2012-01-01

    The properties of a cell’s microenvironment are one of the main driving forces in cellular fate processes and phenotype expression invivo. The ability to create controlled cell microenvironments invitro becomes increasingly important for studying or controlling phenotype expression in tissue engineering and drug discovery applications. This includes the capability to modify material surface properties within well-defined liquid environments in cell culture systems. One successful approach to mimic extra cellular matrix is with porous electrospun polymer fiber scaffolds, while microfluidic networks have been shown to efficiently generate spatially and temporally defined liquid microenvironments. Here, a method to integrate electrospun fibers with microfluidic networks was developed in order to form complex cell microenvironments with the capability to vary relevant parameters. Spatially defined regions of electrospun fibers of both aligned and random orientation were patterned on glass substrates that were irreversibly bonded to microfluidic networks produced in poly-dimethyl-siloxane. Concentration gradients obtained in the fiber containing channels were characterized experimentally and compared with values obtained by computational fluid dynamic simulations. Velocity and shear stress profiles, as well as vortex formation, were calculated to evaluate the influence of fiber pads on fluidic properties. The suitability of the system to support cell attachment and growth was demonstrated with a fibroblast cell line. The potential of the platform was further verified by a functional investigation of neural stem cell alignment in response to orientation of electrospun fibers versus a microfluidic generated chemoattractant gradient of stromal cell-derived factor 1 alpha. The described method is a competitive strategy to create complex microenvironments invitro that allow detailed studies on the interplay of topography, substrate surface properties, and soluble

  9. Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

    PubMed Central

    Li, Liang; Mustafi, Debarshi; Fu, Qiang; Tereshko, Valentina; Chen, Delai L.; Tice, Joshua D.; Ismagilov, Rustem F.

    2006-01-01

    High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a “hybrid” droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as ≈140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in ≈10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 μl of protein solution, ≈1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization. PMID:17159147

  10. Continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging.

    PubMed

    Natarajan, Sriram; Katsamba, Phini S; Miles, Adam; Eckman, Josh; Papalia, Giuseppe A; Rich, Rebecca L; Gale, Bruce K; Myszka, David G

    2008-02-01

    Arraying proteins is often more challenging than creating oligonucleotide arrays. Protein concentration and purity can severely limit the capacity of spots created by traditional pin and ink jet printing techniques. To improve protein printing methods, we have developed a three-dimensional microfluidic system to deposit protein samples within discrete spots (250-microm squares) on a target surface. Our current technology produces a 48-spot array within a 0.5 x 1 cm target area. A chief advantage of this method is that samples may be introduced in continuous flow, which makes it possible to expose each spot to a larger volume of sample than would be possible with standard printing methods. Using Biacore Flexchip (Biacore AB) surface plasmon resonance array-based biosensor as a chip reader, we demonstrate that the microfluidic printer is capable of spotting proteins that are dilute (<0.1 microg/ml) and contain high concentrations of contaminating protein (>10,000-fold molar excess). We also show that the spots created by the microfluidic printer are more uniform and have better-defined borders than what can be achieved with pin printing. The ability to readily print proteins using continuous flow will help expand the application of protein arrays.

  11. Biocompatible patterning of proteins on wettability gradient surface by thermo-transfer printing.

    PubMed

    Kim, Sungho; Ryu, Yong-Sang; Suh, Jeng-Hun; Keum, Chang-Min; Sohn, Youngjoo; Lee, Sin-Doo

    2014-08-01

    We develop a simple and biocompatible method of patterning proteins on a wettability gradient surface by thermo-transfer printing. The wettability gradient is produced on a poly(dimethylsiloxane) (PDMS)-modified glass substrate through the temperature gradient during thermo-transfer printing. The water contact angle on the PDMS-modified surface is found to gradually increase along the direction of the temperature gradient from a low to a high temperature region. Based on the wettability gradient, the gradual change in the adsorption and immobilization of proteins (cholera toxin B subunit) is achieved in a microfluidic cell with the PDMS-modified surface. PMID:25936059

  12. Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

    DOE PAGES

    Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; Doktycz, Mitchel John; Retterer, Scott T.

    2015-12-22

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitatesmore » the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.« less

  13. Towards microfluidic reactors for cell-free protein synthesis at the point-of-care

    SciTech Connect

    Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; Doktycz, Mitchel John; Retterer, Scott T.

    2015-12-22

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro- and nano-fluidic architectures, CFPS can be optimized for point-of care use. Here, we describe the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel reactor and feeder channels. This engineered membrane facilitates the exchange of metabolites, energy, and inhibitory species, prolonging the CFPS reaction and increasing protein yield. Membrane permeability can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. As a result, we show that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor.

  14. Microfluidic three-dimensional hydrodynamic flow focusing for the rapid protein concentration analysis

    PubMed Central

    Hong, Sungmin; Tsou, Pei-Hsiang; Chou, Chao-Kai; Yamaguchi, Hirohito; Su, Chin B.; Hung, Mien-Chie; Kameoka, Jun

    2012-01-01

    A simple microfluidic 3D hydrodynamic flow focusing device has been developed and demonstrated quantitative determinations of quantum dot 525 with antibody (QD525-antibody) and hemagglutinin epitope tagged MAX (HA-MAX) protein concentrations. This device had a step depth cross junction structure at a hydrodynamic flow focusing point at which the analyte stream was flowed into a main detection channel and pinched not only horizontally but also vertically by two sheath streams. As a result, a triangular cross-sectional flow profile of the analyte stream was formed and the laser was focused on the top of the triangular shaped analyte stream. Since the detection volume was smaller than the radius of laser spot, a photon burst histogram showed Gaussian distribution, which was necessary for the quantitative analysis of protein concentration. By using this approach, a linear concentration curve of QD525-antibody down to 10 pM was demonstrated. In addition, the concentration of HA-MAX protein in HEK293 cell lysate was determined as 0.283 ± 0.015 nM. This approach requires for only 1 min determining protein concentration. As the best of our knowledge, this is the first time to determinate protein concentration by using single molecule detection techniques. PMID:23785388

  15. Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures.

    PubMed

    Sikorski, Darek J; Caron, Nicolas J; VanInsberghe, Michael; Zahn, Hans; Eaves, Connie J; Piret, James M; Hansen, Carl L

    2015-10-01

    Heterogeneity in the clonal outputs of individual human embryonic stem cells (hESCs) confounds analysis of their properties in studies of bulk populations and how to manipulate them for clinical applications. To circumvent this problem we developed a microfluidic device that supports the robust generation of colonies derived from single ESCs. This microfluidic system contains 160 individually addressable chambers equipped for perfusion culture of individual hESCs that could be shown to match the growth rates, marker expression and colony morphologies obtained in conventional cultures. Use of this microfluidic device to analyze the clonal growth kinetics of multiple individual hESCs induced to differentiation revealed variable shifts in the growth rate, area per cell and expression of OCT4 in the progeny of individual hESCs. Interestingly, low OCT4 expression, a slower growth rate and low nuclear to cytoplasmic ratios were found to be correlated responses. This study demonstrates how microfluidic systems can be used to enable large scale live-cell imaging of isolated hESCs exposed to changing culture conditions, to examine how different aspects of their variable responses are correlated. PMID:26059045

  16. Toward Microfluidic Reactors for Cell-Free Protein Synthesis at the Point-of-Care.

    PubMed

    Timm, Andrea C; Shankles, Peter G; Foster, Carmen M; Doktycz, Mitchel J; Retterer, Scott T

    2016-02-10

    Cell-free protein synthesis (CFPS) is a powerful technology that allows for optimization of protein production without maintenance of a living system. Integrated within micro and nanofluidic architectures, CFPS can be optimized for point-of-care use. Here, the development of a microfluidic bioreactor designed to facilitate the production of a single-dose of a therapeutic protein, in a small footprint device at the point-of-care, is described. This new design builds on the use of a long, serpentine channel bioreactor and is enhanced by integrating a nanofabricated membrane to allow exchange of materials between parallel "reactor" and "feeder" channels. This engineered membrane facilitates the exchange of metabolites, energy, and inhibitory species, and can be altered by plasma-enhanced chemical vapor deposition and atomic layer deposition to tune the exchange rate of small molecules. This allows for extended reaction times and improved yields. Further, the reaction product and higher molecular weight components of the transcription/translation machinery in the reactor channel can be retained. It has been shown that the microscale bioreactor design produces higher protein yields than conventional tube-based batch formats, and that product yields can be dramatically improved by facilitating small molecule exchange within the dual-channel bioreactor. PMID:26690885

  17. Geometry-induced protein pattern formation.

    PubMed

    Thalmeier, Dominik; Halatek, Jacob; Frey, Erwin

    2016-01-19

    Protein patterns are known to adapt to cell shape and serve as spatial templates that choreograph downstream processes like cell polarity or cell division. However, how can pattern-forming proteins sense and respond to the geometry of a cell, and what mechanistic principles underlie pattern formation? Current models invoke mechanisms based on dynamic instabilities arising from nonlinear interactions between proteins but neglect the influence of the spatial geometry itself. Here, we show that patterns can emerge as a direct result of adaptation to cell geometry, in the absence of dynamical instability. We present a generic reaction module that allows protein densities robustly to adapt to the symmetry of the spatial geometry. The key component is an NTPase protein that cycles between nucleotide-dependent membrane-bound and cytosolic states. For elongated cells, we find that the protein dynamics generically leads to a bipolar pattern, which vanishes as the geometry becomes spherically symmetrical. We show that such a reaction module facilitates universal adaptation to cell geometry by sensing the local ratio of membrane area to cytosolic volume. This sensing mechanism is controlled by the membrane affinities of the different states. We apply the theory to explain AtMinD bipolar patterns in [Formula: see text] EcMinDE Escherichia coli. Due to its generic nature, the mechanism could also serve as a hitherto-unrecognized spatial template in many other bacterial systems. Moreover, the robustness of the mechanism enables self-organized optimization of protein patterns by evolutionary processes. Finally, the proposed module can be used to establish geometry-sensitive protein gradients in synthetic biological systems.

  18. Pattern Recognition of Adsorbing HP Lattice Proteins

    NASA Astrophysics Data System (ADS)

    Wilson, Matthew S.; Shi, Guangjie; Wüst, Thomas; Landau, David P.; Schmid, Friederike

    2015-03-01

    Protein adsorption is relevant in fields ranging from medicine to industry, and the qualitative behavior exhibited by course-grained models could shed insight for further research in such fields. Our study on the selective adsorption of lattice proteins utilizes the Wang-Landau algorithm to simulate the Hydrophobic-Polar (H-P) model with an efficient set of Monte Carlo moves. Each substrate is modeled as a square pattern of 9 lattice sites which attract either H or P monomers, and are located on an otherwise neutral surface. The fully enumerated set of 102 unique surfaces is simulated with each protein sequence. A collection of 27-monomer sequences is used- each of which is non-degenerate and protein-like. Thermodynamic quantities such as the specific heat and free energy are calculated from the density of states, and are used to investigate the adsorption of lattice proteins on patterned substrates. Research supported by NSF.

  19. One-step patterning of hollow microstructures in paper by laser cutting to create microfluidic analytical devices.

    PubMed

    Nie, Jinfang; Liang, Yuanzhi; Zhang, Yun; Le, Shangwang; Li, Dunnan; Zhang, Songbai

    2013-01-21

    In this paper, we report a simple, low-cost method for rapid, highly reproductive fabrication of paper-based microfluidics by using a commercially available, minitype CO(2) laser cutting/engraving machine. This method involves only one operation of cutting a piece of paper by laser according to a predesigned pattern. The hollow microstructures formed in the paper are used as the 'hydrophobic barriers' to define the hydrophilic flowing paths. A typical paper device on a 4 cm × 4 cm piece of paper can be fabricated within ∼7-20 s; it is ready for use once the cutting process is finished. The main fabrication parameters such as the applied current and cutting rate of the laser were optimized. The fabrication resolution and multiplexed analytical capability of the hollow microstructure-patterned paper were also characterized.

  20. Development of an aptamer-based impedimetric bioassay using microfluidic system and magnetic separation for protein detection.

    PubMed

    Wang, Yixian; Ye, Zunzhong; Ping, Jianfeng; Jing, Shunru; Ying, Yibin

    2014-09-15

    An aptamer-based impedimetric bioassay using the microfluidic system and magnetic separation was developed for the sensitive and rapid detection of protein. The microfluidic impedance device was fabricated through integrating the gold interdigitated array microelectrode into a flow cell made of polydimethylsiloxane (PDMS). Aptamer modified magnetic beads were used to capture and separate the target protein, and concentrated into a suitable volume. Then the complexes were injected into the microfluidic flow cell for impedance measurement. To demonstrate the high performance of this novel detection system, thrombin was employed as the target protein. The results showed that the impedance signals at the frequency of 90 kHz have a good linearity with the concentrations of thrombin in a range from 0.1 nM to 10nM and the detection limit is 0.01 nM. Compared with the reported impedimetric aptasensors for thrombin detection, this method possesses several advantages, such as the increasing sensitivity, improving reproducibility, reducing sample volume and assay time. All these demonstrate the proposed detection system is an alternative way to enable sensitive, rapid and specific detection of protein. PMID:24709326

  1. Protein patterns as endpoints in environmental remediation

    SciTech Connect

    Bradley, B.; Brown, D.

    1995-12-31

    Biological endpoints can complement chemical analyses in monitoring environmental remediation. In some cases the levels of chemical detection are so low that the costs of clean-up to no detection would be prohibitive. And chemical tests do not indicate the availability of the contaminants to the biota. On the other hand many if not most biological tests lack specificity. The authors have investigated a protein expression assay to establish an endpoint for clean-up of sulfur mustard and breakdown products. Earthworms (Lumbricus terrestris) were exposed to sulfur mustard (SM), a breakdown product thiodiethanol (TDE), and ethylene glycol, the solvent for the two chemicals. Tissue from the lining of the coelomic cavity was taken from each of 6 worms in each treatment class. Soluble proteins were extracted and separated on one and two-dimensional (1D and 2D) gels. The 1 D gels showed no difference by eye but the patterns from control and solvent control worms on 2D gels differed from those of worms exposed to TDE and SM. The 1D gel data were digitized and analyzed by pattern recognition using artificial neural networks. The protein patterns under the two treatments and the two controls were learned in one set of data and successfully recognized in a second. This indicated that what was learned was useful in recognizing patterns induced by SM and TDE. Thus a possible endpoint for remediation would be the protein pattern at no effect levels of chemicals of interest.

  2. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

    SciTech Connect

    Abdallah, Bahige G.; Zatsepin, Nadia A.; Roy-Chowdhury, Shatabdi; Coe, Jesse; Conrad, Chelsie E.; Dörner, Katerina; Sierra, Raymond G.; Stevenson, Hilary P.; Camacho-Alanis, Fernanda; Grant, Thomas D.; Nelson, Garrett; James, Daniel; Calero, Guillermo; Wachter, Rebekka M.; Spence, John C. H.; Weierstall, Uwe; Fromme, Petra; Ros, Alexandra

    2015-08-19

    We report that the advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ~4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. Ultimately, this method will also

  3. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

    DOE PAGES

    Abdallah, Bahige G.; Zatsepin, Nadia A.; Roy-Chowdhury, Shatabdi; Coe, Jesse; Conrad, Chelsie E.; Dörner, Katerina; Sierra, Raymond G.; Stevenson, Hilary P.; Camacho-Alanis, Fernanda; Grant, Thomas D.; et al

    2015-08-19

    We report that the advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles canmore » be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ~4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. Ultimately, this method

  4. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction

    PubMed Central

    Abdallah, Bahige G.; Zatsepin, Nadia A.; Roy-Chowdhury, Shatabdi; Coe, Jesse; Conrad, Chelsie E.; Dörner, Katerina; Sierra, Raymond G.; Stevenson, Hilary P.; Camacho-Alanis, Fernanda; Grant, Thomas D.; Nelson, Garrett; James, Daniel; Calero, Guillermo; Wachter, Rebekka M.; Spence, John C. H.; Weierstall, Uwe; Fromme, Petra; Ros, Alexandra

    2015-01-01

    The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10–100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to ∼4 Å resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis

  5. Continuous microfluidic DNA and protein trapping and concentration by balancing transverse electrokinetic forces.

    PubMed

    Morales, Mercedes C; Lin, Hao; Zahn, Jeffrey D

    2012-01-01

    Sample pre-concentration can be a critical element to improve sensitivity of integrated microchip assays. In this work a converging Y-inlet microfluidic channel with integrated coplanar electrodes was used to investigate transverse DNA and protein migration under uniform direct current (DC) electric fields to assess the ability to concentrate a sample prior to other enzymatic modifications or capillary electrophoretic separations. Employing a pressure-driven flow to perfuse the microchannel, negatively charged samples diluted in low and high ionic strength buffers were co-infused with a receiving buffer of the same ionic strength into a main daughter channel. Experimental results demonstrated that, depending of the buffer selection, different DNA migration and accumulation dynamics were seen. Charged analytes could traverse the channel width and accumulate at the positive bias electrode in a low electroosmotic mobility, high electrophoretic mobility, high ionic strength buffer or migrated towards an equilibrium position within the channel in a high electroosmotic mobility, high electrophoretic mobility, low ionic strength buffer. The various migration behaviours are the result of a balance between the electrophoretic force and a drag force induced by a recirculating electroosmotic flow generated across the channel width due to the bounding walls. Under continuous flow conditions, DNA samples were concentrated several-fold by balancing these transverse electrokinetic forces. The electrokinetic trapping technique presented here is a simple technique which could be expanded to concentrate or separate other analytes as a preconditioning step for downstream processes. PMID:22045330

  6. Watching Single Enzymes and Fluorescent Proteins in Action in Solution Using a Microfluidic Trap

    NASA Astrophysics Data System (ADS)

    Goldsmith, Randall

    2012-02-01

    Observation of dynamics of single biomolecules over a prolonged time without altering the biomolecule via immobilization is achieved with a specialized microfluidic device. This device, the Anti-Brownian ELectrokinetic (ABEL) Trap, uses real-time electrokinetic feedback to cancel Brownian motion of single objects in solution. First, we use the ABEL Trap to study Allophycocyanin (APC), a photosynthetic antenna-protein and popular fluorescent probe. A complex relationship between fluorescence intensity and lifetime is observed, suggesting light-induced conformational changes and radiative and non-radiative rate fluctuations. Second, we apply the ABEL Trap to single molecules of the multi-copper enzyme blue Nitrite Reductase where a fluorescent label reports on the oxidation state of the Type I Copper. Redox cycling is observed and kinetic analysis allows extraction of the microscopic rate constants in the kinetic scheme. Evidence of a substrate-induced shift of the intramolecular electron transfer rate is seen. Taken together, these observations provide windows of unprecedented detail into the dynamics of solution-phase biomolecules.

  7. High-performance fluorescence-encoded magnetic microbeads as microfluidic protein chip supports for AFP detection.

    PubMed

    Gong, Xiaoqun; Yan, Huan; Yang, Jiumin; Wu, Yudong; Zhang, Jian; Yao, Yingyi; Liu, Ping; Wang, Huiquan; Hu, Zhidong; Chang, Jin

    2016-10-01

    Fluorescence-encoded magnetic microbeads (FEMMs), with the fluorescence encoding ability of quantum dots (QDs) and magnetic enrichment and separation functions of Fe3O4 nanoparticles, have been widely used for multiple biomolecular detection as microfluidic protein chip supports. However, the preparation of FEMMs with long-term fluorescent encoding and immunodetection stability is still a challenge. In this work, we designed a novel high-temperature chemical swelling strategy. The QDs and Fe3O4 nanoparticles were effectively packaged into microbeads via the thermal motion of the polymer chains and the hydrophobic interaction between the nanoparticles and microbeads. The FEMMs obtained a highly uniform fluorescent property and long-term encoding and immunodetection stability and could be quickly magnetically separated and enriched. Then, the QD-encoded magnetic microbeads were applied to alpha fetoprotein (AFP) detection via sandwich immunoreaction. The properties of the encoded microspheres were characterized using a self-designed detecting apparatus, and the target molecular concentration in the sample was also quantified. The results suggested that the high-performance FEMMs have great potential in the field of biomolecular detection.

  8. High-performance fluorescence-encoded magnetic microbeads as microfluidic protein chip supports for AFP detection.

    PubMed

    Gong, Xiaoqun; Yan, Huan; Yang, Jiumin; Wu, Yudong; Zhang, Jian; Yao, Yingyi; Liu, Ping; Wang, Huiquan; Hu, Zhidong; Chang, Jin

    2016-10-01

    Fluorescence-encoded magnetic microbeads (FEMMs), with the fluorescence encoding ability of quantum dots (QDs) and magnetic enrichment and separation functions of Fe3O4 nanoparticles, have been widely used for multiple biomolecular detection as microfluidic protein chip supports. However, the preparation of FEMMs with long-term fluorescent encoding and immunodetection stability is still a challenge. In this work, we designed a novel high-temperature chemical swelling strategy. The QDs and Fe3O4 nanoparticles were effectively packaged into microbeads via the thermal motion of the polymer chains and the hydrophobic interaction between the nanoparticles and microbeads. The FEMMs obtained a highly uniform fluorescent property and long-term encoding and immunodetection stability and could be quickly magnetically separated and enriched. Then, the QD-encoded magnetic microbeads were applied to alpha fetoprotein (AFP) detection via sandwich immunoreaction. The properties of the encoded microspheres were characterized using a self-designed detecting apparatus, and the target molecular concentration in the sample was also quantified. The results suggested that the high-performance FEMMs have great potential in the field of biomolecular detection. PMID:27639146

  9. Different migration patterns of sea urchin and mouse sperm revealed by a microfluidic chemotaxis device.

    PubMed

    Chang, Haixin; Kim, Beum Jun; Kim, Yoon Soo; Suarez, Susan S; Wu, Mingming

    2013-01-01

    Chemotaxis refers to a process whereby cells move up or down a chemical gradient. Sperm chemotaxis is known to be a strategy exploited by marine invertebrates such as sea urchins to reach eggs efficiently in moving water. Less is understood about how or whether chemotaxis is used by mammalian sperm to reach eggs, where fertilization takes place within the confinement of a reproductive tract. In this report, we quantitatively assessed sea urchin and mouse sperm chemotaxis using a recently developed microfluidic model and high-speed imaging. Results demonstrated that sea urchin Arbacia punctulata sperm were chemotactic toward the peptide resact with high chemotactic sensitivity, with an average velocity Vx up the chemical gradient as high as 20% of its average speed (238 μm/s), while mouse sperm displayed no statistically significant chemotactic behavior in progesterone gradients, which had been proposed to guide mammalian sperm toward eggs. This work demonstrates the validity of a microfluidic model for quantitative sperm chemotaxis studies, and reveals a biological insight that chemotaxis up a progesterone gradient may not be a universal strategy for mammalian sperm to reach eggs.

  10. Finding protein-protein interaction patterns by contact map matching.

    PubMed

    Melo, R C; Ribeiro, C; Murray, C S; Veloso, C J M; da Silveira, C H; Neshich, G; Meira, W; Carceroni, R L; Santoro, M M

    2007-01-01

    We propose a novel method for defining patterns of contacts present in protein-protein complexes. A new use of the traditional contact maps (more frequently used for representation of the intra-chain contacts) is presented for analysis of inter-chain contacts. Using an algorithm based on image processing techniques, we can compare protein-protein interaction maps and also obtain a dissimilarity score between them. The same algorithm used to compare the maps can align the contacts of all the complexes and be helpful in the determination of a pattern of conserved interactions at the interfaces. We present an example for the application of this method by analyzing the pattern of interaction of bovine pancreatic trypsin inhibitors and trypsins, chymotrypsins, a thrombin, a matriptase, and a kallikrein - all classified as serine proteases. We found 20 contacts conserved in trypsins and chymotrypsins and 3 specific ones are present in all the serine protease complexes studied. The method was able to identify important contacts for the protein family studied and the results are in agreement with the literature. PMID:18058715

  11. Laser micromachined wax-covered plastic paper as both sputter deposition shadow masks and deep-ultraviolet patterning masks for polymethylmethacrylate-based microfluidic systems

    NASA Astrophysics Data System (ADS)

    Fan, Yiqiang; Li, Huawei; Yi, Ying; Foulds, Ian G.

    2013-10-01

    We report a technically innovative method of fabricating masks for both deep-ultraviolet (UV) patterning and metal sputtering on polymethylmethacrylate (PMMA) for microfluidic systems. We used a CO laser system to cut the required patterns on wax-covered plastic paper; the laser-patterned wax paper will either work as a mask for deep-UV patterning or as a mask for metal sputtering. A microfluidic device was also fabricated to demonstrate the feasibility of this method. The device has two layers: the first layer is a 1-mm thick PMMA substrate that was patterned by deep-UV exposure to create microchannels. The mask used in this process was the laser-cut wax paper. The second layer, also a 1-mm thick PMMA layer, was gold sputtered with patterned wax paper as the shadow mask. These two pieces of PMMA were then bonded to form microchannels with exposed electrodes. This process is a simple and rapid method for creating integrated microfluidic systems that do not require cleanroom facilities.

  12. Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

    PubMed Central

    Blackburn, Matthew C.; Petrova, Ekaterina; Correia, Bruno E.; Maerkl, Sebastian J.

    2016-01-01

    The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3–4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF–DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering. PMID:26704969

  13. Development of a Fast Microfluidic Mixer for Studies of Protein Folding KineticsFinal Report Cover Page

    SciTech Connect

    Bakajin, O

    2005-02-10

    We designed and fabricated mixing devices that will help us elucidate the mechanisms of protein folding through measurements of folding reaction rates. These devices can be used in studying of other biological systems and are compatible with various spectroscopic observation methods. The project involved development of fabrication processes and setup of a laboratory for assembly and characterization of microfluidic devices, as well as measurements of protein folding kinetics. We produced three variants of the mixer: (1) The ultra fast mixer for Foerster Resonance Energy Transfer measurements (described by Anal. Chem. Article UCRL-JRNL-206676) and MicroTAS Conference Proceedings article (UCRL-JC-153057 ) included in the report; (2) The ultra fast mixer for UV measurements (described by the poster presented at MicroTAS conference (UCRL-POST-207476) included in the report); and (3) The mixer for single molecule measurements (described by the Science article UCRL-JC-153057) included in the report. In these mixers, the channels are narrow, ranging from a few to hundreds of {micro}m, so that the flow is laminar and all of the mixing is achieved through diffusion. Our goal is to develop robust microfluidic mixer with at least 100 times lower consumption rate, shorter dead time and time resolution than commercially available mixers that would be compatible with most commonly used spectroscopic methods. We are also developing mixers that can be used in combination with single molecule spectroscopy. The mixers are used to study kinetics of fast protein folding reactions using bulk fluorescence and single molecule fluorescence resonance energy transfer techniques. Capabilities for microfluidic have been developed at BSNL that will be useful for studies of interactions of DNA with proteins and other projects such as the single molecule detector for detection of low concentration of toxins.

  14. Microfluidic Directed Synthesis of Alginate Nanogels with Tunable Pore Size for Efficient Protein Delivery.

    PubMed

    Bazban-Shotorbani, Salime; Dashtimoghadam, Erfan; Karkhaneh, Akbar; Hasani-Sadrabadi, Mohammad Mahdi; Jacob, Karl I

    2016-05-17

    Alginate is a biopolymer with favorable pH-sensitive properties for oral delivery of peptides and proteins. However, conventional alginate nanogels have limitations such as low encapsulation efficiency because of drug leaching during bead preparation and burst release in high pH values. These shortcomings originate from large pore size of the nanogels. In this work, we proposed an on-chip hydrodynamic flow focusing approach for synthesis of alginate nanogels with adjustable pore size to achieve fine-tunable release profile of the encapsulated bioactive agents. It is demonstrated that the microstructure of nanogels can be controlled through adjusting flow ratio and mixing time directed on microfluidic platforms consisting of cross-junction microchannels. In this study, the average pore size of alginate nanogels (i.e., average molecular weight between cross-links, Mc) was related to synthesis parameters. Mc was calculated from equations based on equilibrium swelling theory and proposed methods to modify the theory for pH-sensitive nanogels. In the equations we derived, size and compactness of nanogels are key factors, which can be adjusted by controlling the flow ratio. It was found that increase in flow ratio increases the size of nanogels and decreases their compactness. The size of on-chip generated nanogels for flow ratio of 0.02-0.2 was measured to be in the range of 68-138 nm. Moreover, a method based on the Mie theory was implemented to estimate the aggregation number (Nagg) of polymer chains inside the nanogels as an indicator of compactness. According to the size and compactness results along with equations of modified swelling theory, Mc obtained to be in the range of 0.5-0.8 kDa. The proposed method could be considered as a promising approach for efficient polypeptides encapsulation and their sustained release. PMID:26938744

  15. Microfluidics in biotechnology

    PubMed Central

    Barry, Richard; Ivanov, Dimitri

    2004-01-01

    Microfluidics enables biotechnological processes to proceed on a scale (microns) at which physical processes such as osmotic movement, electrophoretic-motility and surface interactions become enhanced. At the microscale sample volumes and assay times are reduced, and procedural costs are lowered. The versatility of microfluidic devices allows interfacing with current methods and technologies. Microfluidics has been applied to DNA analysis methods and shown to accelerate DNA microarray assay hybridisation times. The linking of microfluidics to protein analysis techologies, e.g. mass spectrometry, enables picomole amounts of peptide to be analysed within a controlled micro-environment. The flexibility of microfluidics will facilitate its exploitation in assay development across multiple biotechnological disciplines. PMID:15056390

  16. An in-line spectrophotometer on a centrifugal microfluidic platform for real-time protein determination and calibration.

    PubMed

    Ding, Zhaoxiong; Zhang, Dongying; Wang, Guanghui; Tang, Minghui; Dong, Yumin; Zhang, Yixin; Ho, Ho-Pui; Zhang, Xuping

    2016-09-21

    In this paper, an in-line, low-cost, miniature and portable spectrophotometric detection system is presented and used for fast protein determination and calibration in centrifugal microfluidics. Our portable detection system is configured with paired emitter and detector diodes (PEDD), where the light beam between both LEDs is collimated with enhanced system tolerance. It is the first time that a physical model of PEDD is clearly presented, which could be modelled as a photosensitive RC oscillator. A portable centrifugal microfluidic system that contains a wireless port in real-time communication with a smartphone has been built to show that PEDD is an effective strategy for conducting rapid protein bioassays with detection performance comparable to that of a UV-vis spectrophotometer. The choice of centrifugal microfluidics offers the unique benefits of highly parallel fluidic actuation at high accuracy while there is no need for a pump, as inertial forces are present within the entire spinning disc and accurately controlled by varying the spinning speed. As a demonstration experiment, we have conducted the Bradford assay for bovine serum albumin (BSA) concentration calibration from 0 to 2 mg mL(-1). Moreover, a novel centrifugal disc with a spiral microchannel is proposed for automatic distribution and metering of the sample to all the parallel reactions at one time. The reported lab-on-a-disc scheme with PEDD detection may offer a solution for high-throughput assays, such as protein density calibration, drug screening and drug solubility measurement that require the handling of a large number of reactions in parallel. PMID:27531134

  17. An in-line spectrophotometer on a centrifugal microfluidic platform for real-time protein determination and calibration.

    PubMed

    Ding, Zhaoxiong; Zhang, Dongying; Wang, Guanghui; Tang, Minghui; Dong, Yumin; Zhang, Yixin; Ho, Ho-Pui; Zhang, Xuping

    2016-09-21

    In this paper, an in-line, low-cost, miniature and portable spectrophotometric detection system is presented and used for fast protein determination and calibration in centrifugal microfluidics. Our portable detection system is configured with paired emitter and detector diodes (PEDD), where the light beam between both LEDs is collimated with enhanced system tolerance. It is the first time that a physical model of PEDD is clearly presented, which could be modelled as a photosensitive RC oscillator. A portable centrifugal microfluidic system that contains a wireless port in real-time communication with a smartphone has been built to show that PEDD is an effective strategy for conducting rapid protein bioassays with detection performance comparable to that of a UV-vis spectrophotometer. The choice of centrifugal microfluidics offers the unique benefits of highly parallel fluidic actuation at high accuracy while there is no need for a pump, as inertial forces are present within the entire spinning disc and accurately controlled by varying the spinning speed. As a demonstration experiment, we have conducted the Bradford assay for bovine serum albumin (BSA) concentration calibration from 0 to 2 mg mL(-1). Moreover, a novel centrifugal disc with a spiral microchannel is proposed for automatic distribution and metering of the sample to all the parallel reactions at one time. The reported lab-on-a-disc scheme with PEDD detection may offer a solution for high-throughput assays, such as protein density calibration, drug screening and drug solubility measurement that require the handling of a large number of reactions in parallel.

  18. DETECTION OF TOPOLOGICAL PATTERNS IN PROTEIN NETWORKS.

    SciTech Connect

    MASLOV,S.SNEPPEN,K.

    2003-11-17

    property of many biological networks that was recently brought to attention of the scientific community [3, 4, 5] is an extremely broad distribution of node connectivities defined as the number of immediate neighbors of a given node in the network. While the majority of nodes have just a few edges connecting them to other nodes in the network, there exist some nodes, that we will refer to as ''hubs'', with an unusually large number of neighbors. The connectivity of the most connected hub in such a network is typically several orders of magnitude larger than the average connectivity in the network. Often the distribution of connectivities of individual nodes can be approximated by a scale-free power law form [3] in which case the network is referred to as scale-free. Among biological networks distributions of node connectivities in metabolic [4], protein interaction [5], and brain functional [6] networks can be reasonably approximated by a power law extending for several orders of magnitude. The set of connectivities of individual nodes is an example of a low-level (single-node) topological property of a network. While it answers the question about how many neighbors a given node has, it gives no information about the identity of those neighbors. It is clear that most functional properties of networks are defined at a higher topological level in the exact pattern of connections of nodes to each other. However, such multi-node connectivity patterns are rather difficult to quantify and compare between networks. In this work we concentrate on multi-node topological properties of protein networks. These networks (as any other biological networks) lack the top-down design. Instead, selective forces of biological evolution shape them from raw material provided by random events such as mutations within individual genes, and gene duplications. As a result their connections are characterized by a large degree of randomness. One may wonder which connectivity patterns are indeed

  19. Determination of cell metabolite VEGF₁₆₅ and dynamic analysis of protein-DNA interactions by combination of microfluidic technique and luminescent switch-on probe.

    PubMed

    Lin, Xuexia; Leung, Ka-Ho; Lin, Ling; Lin, Luyao; Lin, Sheng; Leung, Chung-Hang; Ma, Dik-Lung; Lin, Jin-Ming

    2016-05-15

    In this paper, we rationally design a novel G-quadruplex-selective luminescent iridium (III) complex for rapid detection of oligonucleotide and VEGF165 in microfluidics. This new probe is applied as a convenient biosensor for label-free quantitative analysis of VEGF165 protein from cell metabolism, as well as for studying the kinetics of the aptamer-protein interaction combination with a microfluidic platform. As a result, we have successfully established a quantitative analysis of VEGF165 from cell metabolism. Furthermore, based on the principles of hydrodynamic focusing and diffusive mixing, different transient states during kinetics process were monitored and recorded. Thus, the combination of microfluidic technique and G-quadruplex luminescent probe will be potentially applied in the studies of intramolecular interactions and molecule recognition in the future. PMID:26686922

  20. Microfluidic device, and related methods

    NASA Technical Reports Server (NTRS)

    Wong, Eric W. (Inventor)

    2010-01-01

    A method of making a microfluidic device is provided. The method features patterning a permeable wall on a substrate, and surrounding the permeable wall with a solid, non-permeable boundary structure to establish a microfluidic channel having a cross-sectional dimension less than 5,000 microns and a cross-sectional area at least partially filled with the permeable wall so that fluid flowing through the microfluidic channel at least partially passes through the permeable wall.

  1. Protein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro-tag.

    PubMed

    Lewandowski, Angela T; Yi, Hyunmin; Luo, Xiaolong; Payne, Gregory F; Ghodssi, Reza; Rubloff, Gary W; Bentley, William E

    2008-02-15

    We report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface-assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices. PMID:17625789

  2. Selection of two-phase flow patterns at a simple junction in microfluidic devices.

    PubMed

    Engl, W; Ohata, K; Guillot, P; Colin, A; Panizza, P

    2006-04-01

    We study the behavior of a confined stream made of two immiscible fluids when it reaches a T junction. Two flow patterns are witnessed: the stream is either directed in only one sidearm, yielding a preferential flow pathway for the dispersed phase, or splits between both. We show that the selection of these patterns is not triggered by the shape of the junction nor by capillary effects, but results from confinement. It can be anticipated in terms of the hydrodynamic properties of the flow. A simple model yielding universal behavior in terms of the relevant adimensional parameters of the problem is presented and discussed.

  3. Monitoring the differentiation and migration patterns of neural cells derived from human embryonic stem cells using a microfluidic culture system.

    PubMed

    Lee, Nayeon; Park, Jae Woo; Kim, Hyung Joon; Yeon, Ju Hun; Kwon, Jihye; Ko, Jung Jae; Oh, Seung-Hun; Kim, Hyun Sook; Kim, Aeri; Han, Baek Soo; Lee, Sang Chul; Jeon, Noo Li; Song, Jihwan

    2014-06-01

    Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

  4. Two-Dimensional Protein Patterns in Heterodera glycines

    PubMed Central

    Ferris, V. R.; Ferris, J. M.; Murdock, L. L.

    1985-01-01

    Two-dimensional polyacrylamide gel electrophoretic protein patterns of H. glycines from southern Indiana (Posey County) and northern Indiana (Pulaski County) were largely similar, but many differences existed. The pattern of the Posey isolate was similar to patterns from isolates collected in other areas of the United States. Unique dense protein spots in the pattern of an isolate from Hokkaido, Japan, distinguished it from patterns of six U.S. isolates. PMID:19294120

  5. Protein surface patterning using nanoscale PEG hydrogels.

    PubMed

    Hong, Ye; Krsko, Peter; Libera, Matthew

    2004-12-01

    We have used focused electron-beam cross-linking to create nanosized hydrogels and thus present a new method with which to bring the attractive biocompatibility associated with macroscopic hydrogels into the submicron length-scale regime. Using amine-terminated poly(ethylene glycol) thin films on silicon substrates, we generate nanohydrogels with lateral dimensions of order 200 nm which can swell by a factor of at least five, depending on the radiative dose. With the focused electron beam, high-density arrays of such nanohydrogels can be flexibly patterned onto silicon surfaces. Significantly, the amine groups remain functional after e-beam exposure, and we show that they can be used to covalently bind proteins and other molecules. We use bovine serum albumin to amplify the number of amine groups, and we further demonstrate that different proteins can be covalently bound to different hydrogel pads on the same substrate to create multifunctional surfaces useful in emerging bio/proteomic and sensor technologies.

  6. Suspended microfluidics.

    PubMed

    Casavant, Benjamin P; Berthier, Erwin; Theberge, Ashleigh B; Berthier, Jean; Montanez-Sauri, Sara I; Bischel, Lauren L; Brakke, Kenneth; Hedman, Curtis J; Bushman, Wade; Keller, Nancy P; Beebe, David J

    2013-06-18

    Although the field of microfluidics has made significant progress in bringing new tools to address biological questions, the accessibility and adoption of microfluidics within the life sciences are still limited. Open microfluidic systems have the potential to lower the barriers to adoption, but the absence of robust design rules has hindered their use. Here, we present an open microfluidic platform, suspended microfluidics, that uses surface tension to fill and maintain a fluid in microscale structures devoid of a ceiling and floor. We developed a simple and ubiquitous model predicting fluid flow in suspended microfluidic systems and show that it encompasses many known capillary phenomena. Suspended microfluidics was used to create arrays of collagen membranes, mico Dots (μDots), in a horizontal plane separating two fluidic chambers, demonstrating a transwell platform able to discern collective or individual cellular invasion. Further, we demonstrated that μDots can also be used as a simple multiplexed 3D cellular growth platform. Using the μDot array, we probed the combined effects of soluble factors and matrix components, finding that laminin mitigates the growth suppression properties of the matrix metalloproteinase inhibitor GM6001. Based on the same fluidic principles, we created a suspended microfluidic metabolite extraction platform using a multilayer biphasic system that leverages the accessibility of open microchannels to retrieve steroids and other metabolites readily from cell culture. Suspended microfluidics brings the high degree of fluidic control and unique functionality of closed microfluidics into the highly accessible and robust platform of open microfluidics.

  7. Simple Host−Guest Chemistry To Modulate the Process of Concentration and Crystallization of Membrane Proteins by Detergent Capture in a Microfluidic Device

    SciTech Connect

    Li, Liang; Nachtergaele, Sigrid; Seddon, Annela M.; Tereshko, Valentina; Ponomarenko, Nina; Ismagilov, Rustem F.

    2009-01-15

    This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-{beta}-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.

  8. A novel approach for precisely controlled multiple cell patterning in microfluidic chips by inkjet printing and the detection of drug metabolism and diffusion.

    PubMed

    Zhang, Jie; Chen, Fengming; He, Ziyi; Ma, Yuan; Uchiyama, Katsumi; Lin, Jin-Ming

    2016-05-10

    In this work we report the use of inkjet printing as a precise and convenient means for microscale cell patterning in microfluidic chips followed by cell co-culture, stimulation and analysis. A self-made inkjet printing device was manufactured with adjustable parameters, which was capable of multiple cell printing within biocompatible materials. Sodium alginate was used as a printing matrix for cell encapsulation, and precisely distributed cell arrays on glass slides were obtained by accurate software controlled printing. By covering a PDMS layer with the corresponding microchannels onto the cell array substrate and subsequently injecting an ion cross-linking reagent, the cells containing alginate arrays gelated immediately and were immobilized on the bottom of the microchip, which could be utilized for cell culture and analysis. HepG2 cells and U251 cells were successfully co-patterned in the microchip and used for drug metabolism and diffusion experiment to imitate the in vivo situation, as a means to ascertain the capability of the system for precise microscale cell patterning in a microchip. The prodrug tegafur was metabolized by HepG2 cells into the active anticancer compound 5-fluorouracil and this produced an adverse gradient effect on U251 cells according to the distance from the HepG2 cells. The developed approach presented a feasible way to integrate inkjet cell printing and microfluidic chips for the first time, which is proved to be capable of spatially controlled printing of multiple kinds of cells into a microchip for cell culture, stimulation and analysis, which could be applied to tissue engineering, drug testing and related areas. We envision that the approach will help significantly increase the cell patterning efficacy in microfluidic chips as well as reduce the extent of laborious experimental work. PMID:27045202

  9. Microfluidic electronics.

    PubMed

    Cheng, Shi; Wu, Zhigang

    2012-08-21

    Microfluidics, a field that has been well-established for several decades, has seen extensive applications in the areas of biology, chemistry, and medicine. However, it might be very hard to imagine how such soft microfluidic devices would be used in other areas, such as electronics, in which stiff, solid metals, insulators, and semiconductors have previously dominated. Very recently, things have radically changed. Taking advantage of native properties of microfluidics, advances in microfluidics-based electronics have shown great potential in numerous new appealing applications, e.g. bio-inspired devices, body-worn healthcare and medical sensing systems, and ergonomic units, in which conventional rigid, bulky electronics are facing insurmountable obstacles to fulfil the demand on comfortable user experience. Not only would the birth of microfluidic electronics contribute to both the microfluidics and electronics fields, but it may also shape the future of our daily life. Nevertheless, microfluidic electronics are still at a very early stage, and significant efforts in research and development are needed to advance this emerging field. The intention of this article is to review recent research outcomes in the field of microfluidic electronics, and address current technical challenges and issues. The outlook of future development in microfluidic electronic devices and systems, as well as new fabrication techniques, is also discussed. Moreover, the authors would like to inspire both the microfluidics and electronics communities to further exploit this newly-established field.

  10. Automatic disease screening method using image processing for dried blood microfluidic drop stain pattern recognition.

    PubMed

    Sikarwar, Basant S; Roy, Mukesh; Ranjan, Priya; Goyal, Ayush

    2016-07-01

    This paper examines programmed automatic recognition of infection from samples of dried stains of micro-scale drops of patient blood. This technique has the upside of being low-cost and less-intrusive and not requiring puncturing the patient with a needle for drawing blood, which is especially critical for infants and the matured. It also does not require expensive pathological blood test laboratory equipment. The method is shown in this work to be successful for ailment identification in patients suffering from tuberculosis and anaemia. Illness affects the physical properties of blood, which thus influence the samples of dried micro-scale blood drop stains. For instance, if a patient has a severe drop in platelet count, which is often the case of dengue or malaria patients, the blood's physical property of viscosity drops substantially, i.e. the blood is thinner. Thus, the blood micro-scale drop stain samples can be utilised for diagnosing maladies. This paper presents programmed automatic examination of the dried micro-scale drop blood stain designs utilising an algorithm based on pattern recognition. The samples of micro-scale blood drop stains of ordinary non-infected people are clearly recognisable as well as the samples of micro-scale blood drop stains of sick people, due to key distinguishing features. As a contextual analysis, the micro-scale blood drop stains of patients infected with tuberculosis have been contrasted with the micro-scale blood drop stains of typical normal healthy people. The paper dives into the fundamental flow mechanics behind how the samples of the dried micro-scale blood drop stain is shaped. What has been found is a thick ring like feature in the dried micro-scale blood drop stains of non-ailing people and thin shape like lines in the dried micro-scale blood drop stains of patients with anaemia or tuberculosis disease. The ring like feature at the periphery is caused by an outward stream conveying suspended particles to the edge

  11. Surface acoustic wave microfluidics

    PubMed Central

    Ding, Xiaoyun; Li, Peng; Lin, Sz-Chin Steven; Stratton, Zackary S.; Nama, Nitesh; Guo, Feng; Slotcavage, Daniel; Mao, Xiaole; Shi, Jinjie; Costanzo, Francesco; Huang, Tony Jun

    2014-01-01

    The recent introduction of surface acoustic wave (SAW) technology onto lab-on-a-chip platforms has opened a new frontier in microfluidics. The advantages provided by such SAW microfluidics are numerous: simple fabrication, high biocompatibility, fast fluid actuation, versatility, compact and inexpensive devices and accessories, contact-free particle manipulation, and compatibility with other microfluidic components. We believe that these advantages enable SAW microfluidics to play a significant role in a variety of applications in biology, chemistry, engineering, and medicine. In this review article, we discuss the theory underpinning SAWs and their interactions with particles and the contacting fluids in which they are suspended. We then review the SAW-enabled microfluidic devices demonstrated to date, starting with devices that accomplish fluid mixing and transport through the use of travelling SAW; we follow that by reviewing the more recent innovations achieved with standing SAW that enable such actions as particle/cell focusing, sorting, and patterning. Finally, we look forward and appraise where the discipline of SAW microfluidics could go next. PMID:23900527

  12. Surface acoustic wave microfluidics.

    PubMed

    Ding, Xiaoyun; Li, Peng; Lin, Sz-Chin Steven; Stratton, Zackary S; Nama, Nitesh; Guo, Feng; Slotcavage, Daniel; Mao, Xiaole; Shi, Jinjie; Costanzo, Francesco; Huang, Tony Jun

    2013-09-21

    The recent introduction of surface acoustic wave (SAW) technology onto lab-on-a-chip platforms has opened a new frontier in microfluidics. The advantages provided by such SAW microfluidics are numerous: simple fabrication, high biocompatibility, fast fluid actuation, versatility, compact and inexpensive devices and accessories, contact-free particle manipulation, and compatibility with other microfluidic components. We believe that these advantages enable SAW microfluidics to play a significant role in a variety of applications in biology, chemistry, engineering and medicine. In this review article, we discuss the theory underpinning SAWs and their interactions with particles and the contacting fluids in which they are suspended. We then review the SAW-enabled microfluidic devices demonstrated to date, starting with devices that accomplish fluid mixing and transport through the use of travelling SAW; we follow that by reviewing the more recent innovations achieved with standing SAW that enable such actions as particle/cell focusing, sorting and patterning. Finally, we look forward and appraise where the discipline of SAW microfluidics could go next.

  13. Control of crystal polymorph in microfluidics using molluscan 28 kDa Ca²(+)-binding protein.

    PubMed

    Ji, Bozhi; Cusack, Maggie; Freer, Andy; Dobson, Phil S; Gadegaard, Nikolaj; Yin, Huabing

    2010-10-01

    Biominerals produced by biological systems in physiologically relevant environments possess extraordinary properties that are often difficult to replicate under laboratory conditions. Understanding the mechanism that underlies the process of biomineralisation can lead to novel strategies in the development of advanced materials. Using microfluidics, we have demonstrated for the first time, that an extrapallial (EP) 28 kDa protein, located in the extrapallial compartment between mantle and shell of Mytilus edulis, can influence, at both micro- and nanoscopic levels, the morphology, structure and polymorph that is laid down in the shell ultrastructure. Crucially, this influence is predominantly dependent on the existence of an EP protein concentration gradient and its consecutive interaction with Ca²(+) ions. Novel lemon-shaped hollow vaterite structures with a clearly defined nanogranular assembly occur only where particular EP protein and Ca²(+) gradients co-exist. Computational fluid dynamics enabled the progress of the reaction to be mapped and the influence of concentration gradients across the device to be calculated. Importantly, these findings could not have been observed using conventional bulk mixing methods. Our findings not only provide direct experimental evidence of the potential influence of EP proteins in crystal formation, but also offer a new biomimetic strategy to develop functional biomaterials for applications such as encapsulation and drug delivery.

  14. Label-free detection of DNA-binding proteins based on microfluidic solid-state molecular beacon sensor.

    PubMed

    Wang, Jun; Onoshima, Daisuke; Aki, Michihiko; Okamoto, Yukihiro; Kaji, Noritada; Tokeshi, Manabu; Baba, Yoshinobu

    2011-05-01

    A solid-state molecular beacon using a gold support as a fluorescence quencher is combined with a polydimethylsiloxane (PDMS) microfluidic channel to construct an optical sensor for detecting single-stranded DNA binding protein (SSBP) and histone protein. The single-stranded DNA-Cy3 probe or double-stranded DNA-Cy3 probe immobilized on the gold surface is prepared for the detection of SSBP or histone, respectively. Due to the different quenching ability of gold to the immobilized single-stranded DNA-Cy3 probe and the immobilized double-stranded DNA-Cy3 probe, low fluorescence intensity of the attached single-stranded DNA-Cy3 is obtained in SSBP detection, whereas high fluorescence intensity of the attached double-stranded DNA-Cy3 is obtained in histone detection. The amounts of SSBP in sample solutions are determined from the degree of fluorescence recovery of the immobilized single-stranded DNA-Cy3 probe, whereas that of histone in sample solutions is determined from the degree of fluorescence quenching of the immobilized double-stranded DNA-Cy3 probe. Using this approach, label-free detection of target proteins at nanomolar concentrations is achieved in a convenient, general, continuous flow format. Our approach has high potential for the highly sensitive label-free detection of various proteins based on binding-induced conformation changes of immobilized DNA probes.

  15. Microfluidic fuel cells

    NASA Astrophysics Data System (ADS)

    Kjeang, Erik

    Microfluidic fuel cell architectures are presented in this thesis. This work represents the mechanical and microfluidic portion of a microfluidic biofuel cell project. While the microfluidic fuel cells developed here are targeted to eventual integration with biocatalysts, the contributions of this thesis have more general applicability. The cell architectures are developed and evaluated based on conventional non-biological electrocatalysts. The fuel cells employ co-laminar flow of fuel and oxidant streams that do not require a membrane for physical separation, and comprise carbon or gold electrodes compatible with most enzyme immobilization schemes developed to date. The demonstrated microfluidic fuel cell architectures include the following: a single cell with planar gold electrodes and a grooved channel architecture that accommodates gaseous product evolution while preventing crossover effects; a single cell with planar carbon electrodes based on graphite rods; a three-dimensional hexagonal array cell based on multiple graphite rod electrodes with unique scale-up opportunities; a single cell with porous carbon electrodes that provides enhanced power output mainly attributed to the increased active area; a single cell with flow-through porous carbon electrodes that provides improved performance and overall energy conversion efficiency; and a single cell with flow-through porous gold electrodes with similar capabilities and reduced ohmic resistance. As compared to previous results, the microfluidic fuel cells developed in this work show improved fuel cell performance (both in terms of power density and efficiency). In addition, this dissertation includes the development of an integrated electrochemical velocimetry approach for microfluidic devices, and a computational modeling study of strategic enzyme patterning for microfluidic biofuel cells with consecutive reactions.

  16. Electro-Microfluidic Packaging

    SciTech Connect

    BENAVIDES, GILBERT L.; GALAMBOS, PAUL C.

    2002-06-01

    Electro-microfluidics is experiencing explosive growth in new product developments. There are many commercial applications for electro-microfluidic devices such as chemical sensors, biological sensors, and drop ejectors for both printing and chemical analysis. The number of silicon surface micromachined electro-microfluidic products is likely to increase. Manufacturing efficiency and integration of microfluidics with electronics will become important. Surface micromachined microfluidic devices are manufactured with the same tools as IC's (integrated circuits) and their fabrication can be incorporated into the IC fabrication process. In order to realize applications for devices must be developed. An Electro-Microfluidic Dual In-line Package (EMDIP{trademark}) was developed surface micromachined electro-microfluidic devices, a practical method for getting fluid into these to be a standard solution that allows for both the electrical and the fluidic connections needed to operate a great variety of electro-microfluidic devices. The EMDIP{trademark} includes a fan-out manifold that, on one side, mates directly with the 200 micron diameter Bosch etched holes found on the device, and, on the other side, mates to lager 1 mm diameter holes. To minimize cost the EMDIP{trademark} can be injection molded in a great variety of thermoplastics which also serve to optimize fluid compatibility. The EMDIP{trademark} plugs directly into a fluidic printed wiring board using a standard dual in-line package pattern for the electrical connections and having a grid of multiple 1 mm diameter fluidic connections to mate to the underside of the EMDIP{trademark}.

  17. Microfluidic beads-based immunosensor for sensitive detection of cancer biomarker proteins using multienzyme-nanoparticle amplification and quantum dots labels.

    PubMed

    Zhang, He; Liu, Lian; Fu, Xin; Zhu, Zhenjun

    2013-04-15

    This study reports the development of a microfluidic beads-based immunosensor for sensitive detection of cancer biomarker α-fetoprotein (AFP) that uses multienzyme-nanoparticle amplification and quantum dots labels. This method utilizes microbeads functionalized with the capture antibodies (Ab₁) and modified electron rich proteins as sensing platform that was fabricated within a microfluidic channel, and uses gold nanoparticles (AuNPs) functionalized with the horseradish peroxidase (HRP) and the detection antibodies (Ab₂) as label. Greatly enhanced sensitivity for the cancer biomarker is based on a dual signal amplification strategy: first, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Enhanced sensitivity was achieved by introducing the multi-HRP-antibody functionalized AuNPs onto the surface of microbeads through "sandwich" immunoreactions and subsequently multiple biotin moieties could be deposited onto the surface of beads resulted from the oxidation of biotin-tyramine by hydrogen peroxide. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Secondly, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro-channel delivers fresh analyte solution to the reaction site which maintains a high concentration gradient differential to enhance mass transport. Based on the dual signal amplification strategy, the developed microfluidic bead-based immunosensor could discriminate as low as 0.2 pg mL⁻¹ AFP in 10 μL of undiluted calf serum (0.2 fg/chip), and showed a 500-fold increase in detection limit compared to the off-chip test and 50-fold increase in detection limit compared to microfluidic beads-based immunoassay using single label HRP-Ab₂. The immunosensor showed acceptable repeatability and reproducibility. This microfluidic beads

  18. Network based approaches reveal clustering in protein point patterns

    NASA Astrophysics Data System (ADS)

    Parker, Joshua; Barr, Valarie; Aldridge, Joshua; Samelson, Lawrence E.; Losert, Wolfgang

    2014-03-01

    Recent advances in super-resolution imaging have allowed for the sub-diffraction measurement of the spatial location of proteins on the surfaces of T-cells. The challenge is to connect these complex point patterns to the internal processes and interactions, both protein-protein and protein-membrane. We begin analyzing these patterns by forming a geometric network amongst the proteins and looking at network measures, such the degree distribution. This allows us to compare experimentally observed patterns to models. Specifically, we find that the experimental patterns differ from heterogeneous Poisson processes, highlighting an internal clustering structure. Further work will be to compare our results to simulated protein-protein interactions to determine clustering mechanisms.

  19. PDMS bonding to a bio-friendly photoresist via self-polymerized poly(dopamine) adhesive for complex protein micropatterning inside microfluidic channels.

    PubMed

    Kim, Miju; Song, Kwang Hoon; Doh, Junsang

    2013-12-01

    Protein micropatterned surfaces integrated with microfluidics are useful in numerous bioanalytical and biological applications. In this study, we demonstrated the fabrication of complex protein micropatterned surfaces within poly(dimethylsiloxane) (PDMS) microfluidic channels by attaching the PDMS channels to bio-friendly photoresist films and subsequently performing microscope projection photolithography (MPP). A muscle-inspired poly(dopamine) (PDA) coating was employed to mediate the bonding between the PDMS and the bio-friendly photoresist poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate) (PDMP). By adjusting the dip-coating time for the PDA coating, we could successfully introduce sufficient amounts of functional groups on the PDMP surfaces to mediate strong bonding between the PDMS channels and the PDA-coated PDMP thin films with minimal alteration of the surface properties of the PDMP thin films that are critical for protein micropatterning. Using this novel bonding strategy, we successfully fabricated multiple protein micropatterns and gradient micropatterns of proteins within microfluidic channels. The technique developed in this study will be useful for the fabrication of complex biochips for multiplex bioassays and fundamental cell biological studies.

  20. Microfluidic Diffusion Analysis of the Sizes and Interactions of Proteins under Native Solution Conditions.

    PubMed

    Arosio, Paolo; Müller, Thomas; Rajah, Luke; Yates, Emma V; Aprile, Francesco A; Zhang, Yingbo; Cohen, Samuel I A; White, Duncan A; Herling, Therese W; De Genst, Erwin J; Linse, Sara; Vendruscolo, Michele; Dobson, Christopher M; Knowles, Tuomas P J

    2016-01-26

    Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space-time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures.

  1. Wax-bonding 3D microfluidic chips.

    PubMed

    Gong, Xiuqing; Yi, Xin; Xiao, Kang; Li, Shunbo; Kodzius, Rimantas; Qin, Jianhua; Wen, Weijia

    2010-10-01

    We report a simple, low-cost and detachable microfluidic chip incorporating easily accessible paper, glass slides or other polymer films as the chip materials along with adhesive wax as the recycling bonding material. We use a laser to cut through the paper or film to form patterns and then sandwich the paper and film between glass sheets or polymer membranes. The hot-melt adhesive wax can realize bridge bonding between various materials, for example, paper, polymethylmethacrylate (PMMA) film, glass sheets, or metal plate. The bonding process is reversible and the wax is reusable through a melting and cooling process. With this process, a three-dimensional (3D) microfluidic chip is achievable by vacuating and venting the chip in a hot-water bath. To study the biocompatibility and applicability of the wax-based microfluidic chip, we tested the PCR compatibility with the chip materials first. Then we applied the wax-paper based microfluidic chip to HeLa cell electroporation (EP). Subsequently, a prototype of a 5-layer 3D chip was fabricated by multilayer wax bonding. To check the sealing ability and the durability of the chip, green fluorescence protein (GFP) recombinant Escherichia coli (E. coli) bacteria were cultured, with which the chemotaxis of E. coli was studied in order to determine the influence of antibiotic ciprofloxacin concentration on the E. coli migration.

  2. Spectroscopic Analysis of Red Fluorescent Proteins and Development of a Microfluidic Cell Sorter for the Generation of Improved Variants

    NASA Astrophysics Data System (ADS)

    Lubbeck, Jennifer L.

    The discovery of the green fluorescent protein (GFP) launched the development of a wide variety of fluorescent protein (FP) mutants whose spectral and photophysical diversity revolutionized in vivo imaging. The excitation and emission spectra of red fluorescent proteins (RFPs), in particular, have been ideally tuned to a window optically favorable for in vivo work. However, their quantum yields, photostabilities and fluorescence intermittency properties require improvement if they are to be broadly employed for low-copy or single-molecule measurements. Attempts to engineer improved RFPs often result in optimization of one photophysical property at the expense of others. We developed a microfluidic-based cytometer for screening HeLa cell-based genetic RFP-libraries simultaneously on the basis of fluorescence lifetime (a proxy for quantum yield), photostability, and brightness. Ten 532 nm excitation beams interrogate each cell in flow. The first is electro-optically modulated (30 MHz) to enable lifetime measurement with phase fluorimetry. The remaining beams act as a pulse sequence for isolating the irreversible photobleaching time constant. Optical-force switching is employed to sort cells based on any combination of the photophysical parameters. Screening with this instrument enables identification of regions of the structure that synergistically affect quantum yield and photostability and the sorting capability provides a new tool for accelerating the development of next generation RFPs.

  3. Fabrication of a gel particle array in a microfluidic device for bioassays of protein and glucose in human urine samples.

    PubMed

    Lin, Ling; Gao, Zhaoxin; Wei, Huibin; Li, Haifang; Wang, Feng; Lin, Jin-Ming

    2011-09-01

    This paper describes a simple method for fabricating a series of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures inside microfluidic channels as probe for proteins and glucose. In order to demonstrate the feasibility of this newly developed system, bovine serum albumin (BSA) was chosen as a model protein. PEG microcolumns were used for the parallel detection of multiple components. Using tetrabromophenol blue (TBPB) and the horseradish peroxidase/glucose oxidase reaction system, bovine serum albumin (BSA) and glucose in human urine were detected by color changes. The color changes for BSA within a concentration range of 1-150 μM, and glucose within a range of 50 mM-2 M could be directly distinguished by eyes or precisely identified by optical microscope. To show the practicability of the gel particle array, protein and glucose concentrations of real human urine samples were determined, resulting in a good correlation with hospital analysis. Notably, only a 5 µL sample was needed for a parallel measurement of both analytes. Conveniently, no special readout equipment or power source was required during the diagnosis process, which is promising for an application in rapid point-of-care diagnosis. PMID:22662039

  4. Microfluidics experiments of dissolution in a fracture. Influence of Damköhler and Péclet numbers, and of the geometry on the dissolution pattern

    NASA Astrophysics Data System (ADS)

    Osselin, Florian; Budek, Agnieszka; Cybulski, Olgierd; Szymczak, Piotr

    2015-04-01

    Dissolution of natural rocks is an ever present phenomenon in nature. The shaping of natural landscapes by the dissolution of limestone gives for example birth to exceptional features like karsts. Currently dissolution is also at the heart of key research topics as Carbon Capture and Storage or Enhanced Oil Recovery. The basics principles of dissolution are well-known, however, the sheer amount of different patterns arising from these mechanisms and the strong dependency on parameters such as pore network, chemical composition and flow rate, make it particularly difficult to study theoretically and experimentally. In this study we present a microfluidic experiment simulating the behavior of a dissolving fluid in a fracture. The experiments consist of a chip of gyspum inserted between two polycarbonate plates and subjected to a constant flow rate of pure water. The point in using microfluidics is that it allows a complete control on the experimental parameters such as geometry and chemical composition of the porous medium, flow rate, fracture aperture, roughness of the fracture walls, and an in situ observation of the geometry evolution which is impossible with 3D natural rocks. Thanks to our experiments we have been able to cover the whole range of dissolution patterns, from wormholing or DLA fingering to homogeneous dissolution, by changing Péclet and Damköhler numbers. Moreover, we have been able to tweak the geometry of our artificial fracture, inserting finger seeds or non-dissolvable obstacles. The comparison of the experimental patterns with the numerical dissolution code dissol (Szymczak and Ladd 2011) has then shown a very good correlation of the patterns, giving confidence in both experiments and modeling.

  5. Encoding protein-ligand interaction patterns in fingerprints and graphs.

    PubMed

    Desaphy, Jérémy; Raimbaud, Eric; Ducrot, Pierre; Rognan, Didier

    2013-03-25

    We herewith present a novel and universal method to convert protein-ligand coordinates into a simple fingerprint of 210 integers registering the corresponding molecular interaction pattern. Each interaction (hydrophobic, aromatic, hydrogen bond, ionic bond, metal complexation) is detected on the fly and physically described by a pseudoatom centered either on the interacting ligand atom, the interacting protein atom, or the geometric center of both interacting atoms. Counting all possible triplets of interaction pseudoatoms within six distance ranges, and pruning the full integer vector to keep the most frequent triplets enables the definition of a simple (210 integers) and coordinate frame-invariant interaction pattern descriptor (TIFP) that can be applied to compare any pair of protein-ligand complexes. TIFP fingerprints have been calculated for ca. 10,000 druggable protein-ligand complexes therefore enabling a wide comparison of relationships between interaction pattern similarity and ligand or binding site pairwise similarity. We notably show that interaction pattern similarity strongly depends on binding site similarity. In addition to the TIFP fingerprint which registers intermolecular interactions between a ligand and its target protein, we developed two tools (Ishape, Grim) to align protein-ligand complexes from their interaction patterns. Ishape is based on the overlap of interaction pseudoatoms using a smooth Gaussian function, whereas Grim utilizes a standard clique detection algorithm to match interaction pattern graphs. Both tools are complementary and enable protein-ligand complex alignments capitalizing on both global and local pattern similarities. The new fingerprint and companion alignment tools have been successfully used in three scenarios: (i) interaction-biased alignment of protein-ligand complexes, (ii) postprocessing docking poses according to known interaction patterns for a particular target, and (iii) virtual screening for bioisosteric

  6. Directed self-assembly of proteins into discrete radial patterns

    PubMed Central

    Thakur, Garima; Prashanthi, Kovur; Thundat, Thomas

    2013-01-01

    Unlike physical patterning of materials at nanometer scale, manipulating soft matter such as biomolecules into patterns is still in its infancy. Self-assembled monolayer (SAM) with surface density gradient has the capability to drive biomolecules in specific directions to create hierarchical and discrete structures. Here, we report on a two-step process of self-assembly of the human serum albumin (HSA) protein into discrete ring structures based on density gradient of SAM. The methodology involves first creating a 2-dimensional (2D) polyethylene glycol (PEG) islands with responsive carboxyl functionalities. Incubation of proteins on such pre-patterned surfaces results in direct self-assembly of protein molecules around PEG islands. Immobilization and adsorption of protein on such structures over time evolve into the self-assembled patterns. PMID:23719678

  7. Selective memory generalization by spatial patterning of protein synthesis

    PubMed Central

    O’Donnell, Cian; Sejnowski, Terrence J.

    2014-01-01

    Summary Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings we proposed a novel two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. PMID:24742462

  8. High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking

    PubMed Central

    Ullman, G.; Wallden, M.; Marklund, E. G.; Mahmutovic, A.; Razinkov, Ivan; Elf, J.

    2013-01-01

    We have developed a method combining microfluidics, time-lapsed single-molecule microscopy and automated image analysis allowing for the observation of an excess of 3000 complete cell cycles of exponentially growing Escherichia coli cells per experiment. The method makes it possible to analyse the rate of gene expression at the level of single proteins over the bacterial cell cycle. We also demonstrate that it is possible to count the number of non-specifically DNA binding LacI–Venus molecules using short excitation light pulses. The transcription factors are localized on the nucleoids in the cell and appear to be uniformly distributed on chromosomal DNA. An increase in the expression of LacI is observed at the beginning of the cell cycle, possibly because some gene copies are de-repressed as a result of partitioning inequalities at cell division. Finally, a size–growth rate uncertainty relation is observed where cells living in rich media vary more in the length at birth than in generation time, and the opposite is true for cells living in poorer media. PMID:23267179

  9. Detection of Streptavidin-Biotin Protein Complexes Using Three-Dimensional MOSFET in the Si Micro-Fluidic Channel

    NASA Astrophysics Data System (ADS)

    Han, Dae-Il; Kim, Dong-Sun; Park, Jee-Eun; Shin, Jang-Kyoo; Kong, Seong-Ho; Choi, Pyung; Lee, Jong-Hyun; Lim, Geunbae

    2005-07-01

    To detect the electrical reaction characteristics of streptavidin-biotin protein complexes, a three-dimensional (3-D) p-channel metal oxide semiconductor field effect transistor (PMOSFET)-type biosensor was fabricated in the convex corner of a micro-fluidic channel by tetramethyl ammonium hydroxide (TMAH) anisotropic etching. Au, which has a chemical affinity with thiol, was used as the gate metal for immobilizing a self-assembled monolayer (SAM). The SAM was used to immobilize streptavidin. The hydroxyl group of SAM was bound with the amine group of streptavidin. After that, streptavidin and biotin were bound by their high affinity (Ka˜ 1015 Mol-1). The measurements were performed in phosphate-buffered saline (PBS; pH 6.4, 20 mM) solution. Ag/AgCl was used as the reference electrode. The bindings of SAM, streptavidin and biotin caused a variation in the drain current of the 3-D MOSFET. To verify the interaction among the SAM, streptavidin and biotin, surface plasmon resonance (SPR) measurement was performed.

  10. Ultrasensitive Detection of Low-Abundance Surface-Marker Protein using Isothermal Rolling Circle Amplification in Microfluidic Nano-Liter Platform

    PubMed Central

    Konry, Tania; Yarmush, Joel M.; Irimia, Daniel

    2011-01-01

    With advances in immunology and cancer biology, there is an unmet need for increasingly sensitive systems to monitor the expression of specific cell markers for the development of new diagnostic and therapeutic tools. To address this challenge, we have applied a highly sensitive labeling method that translates antigen-antibody recognition processes into DNA detection event that can be greatly amplified via isothermal Rolling Circle Amplification (RCA). By merging the single-molecule detection power of RCA reaction with microfluidic technology we were able to demonstrate that identification of specific protein markers can be achieved on tumor cell surface in miniaturized nano-liter reaction droplets. Furthermore, this combined approach of signal amplification in a microfluidic format could extend the utility of existing methods by reducing sample and reagent consumption and enhancing the sensitivities and specificities for various applications, including early diagnosis of cancer. PMID:21294269

  11. Elastohydrodynamics and Kinetics of Protein Patterning in the Immunological Synapse

    PubMed Central

    Carlson, Andreas; Mahadevan, L.

    2015-01-01

    We propose a minimal mathematical model for the physical basis of membrane protein patterning in the immunological synapse (IS), which encompass membrane mechanics, protein binding kinetics and motion, and fluid flow in the synaptic cleft. Our theory leads to simple predictions for the spatial and temporal scales of protein cluster formation, growth and arrest as a function of membrane stiffness, rigidity and kinetics of the adhesive proteins, and the fluid flow in the synaptic cleft. Numerical simulations complement these scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment shows that passive elastohydrodynamics and kinetics of protein binding in the synaptic cleft can describe the short-time formation and organization of protein clusters, without evoking any active processes in the cytoskeleton. Despite the apparent complexity of the process, our analysis shows that just two dimensionless parameters characterize the spatial and temporal evolution of the protein pattern: a ratio of membrane elasticity to protein stiffness, and the ratio of a hydrodynamic time scale for fluid flow relative to the protein binding rate. A simple phase diagram encompasses the variety of patterns that can arise. PMID:26699430

  12. Elastohydrodynamics and Kinetics of Protein Patterning in the Immunological Synapse.

    PubMed

    Carlson, Andreas; Mahadevan, L

    2015-12-01

    We propose a minimal mathematical model for the physical basis of membrane protein patterning in the immunological synapse (IS), which encompass membrane mechanics, protein binding kinetics and motion, and fluid flow in the synaptic cleft. Our theory leads to simple predictions for the spatial and temporal scales of protein cluster formation, growth and arrest as a function of membrane stiffness, rigidity and kinetics of the adhesive proteins, and the fluid flow in the synaptic cleft. Numerical simulations complement these scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment shows that passive elastohydrodynamics and kinetics of protein binding in the synaptic cleft can describe the short-time formation and organization of protein clusters, without evoking any active processes in the cytoskeleton. Despite the apparent complexity of the process, our analysis shows that just two dimensionless parameters characterize the spatial and temporal evolution of the protein pattern: a ratio of membrane elasticity to protein stiffness, and the ratio of a hydrodynamic time scale for fluid flow relative to the protein binding rate. A simple phase diagram encompasses the variety of patterns that can arise. PMID:26699430

  13. Excimer laser ablation for spatially controlled protein patterns

    NASA Astrophysics Data System (ADS)

    Thissen, Helmut; Hayes, Jason P.; Kingshott, Peter; Johnson, Graham; Harvey, Erol C.; Griesser, Hans J.

    2001-11-01

    Two-dimensional control over the location of proteins on surfaces is desired for a number of applications including diagnostic tests and tissue engineered medical devices. Many of these applications require patterns of specific proteins that allow subsequent two-dimensionally controlled cell attachment. The ideal technique would allow the deposition of specific protein patterns in areas where cell attachment is required, with complete prevention of unspecific protein adsorption in areas where cells are not supposed to attach. In our study, collagen I was used as an example for an extracellular matrix protein known to support the attachment of bovine corneal epithelial cells. An allylamine plasma polymer was deposited on a silicon wafer substrate, followed by grafting of poly(ethylene oxide). Two-dimensional control over the surface chemistry was achieved using a 248 nm excimer laser. Results obtained by XPS and AFM show that the combination of extremely low-fouling surfaces with excimer laser ablation can be used effectively for the production of spatially controlled protein patterns with a resolution of less than 1 micrometers . Furthermore, it was shown that bovine corneal epithelial cell attachment followed exactly the created protein patterns. The presented method is an effective tool for a number of in vitro and in vivo applications.

  14. Protein patterning: a comparison of direct spotting versus microcontact printing

    NASA Astrophysics Data System (ADS)

    Clancy, Kathryn F. A.; Nicolau, Dan V.

    2015-03-01

    Protein microarrays are used various research areas including drug discovery, diagnosis, and analysis of protein-ligand interactions. Their efficacy depends on a well-defined pattern of immobilized proteins that also have retained their bioactivity. Protein microarrays are classically fabricated using the robotic spotting drop method ("pin printing"), which can lead to spots with uneven protein concentration within the spotted area, leading to difficult to quantify readings. Among the alternative techniques, microcontact printing (μCP) with a poly(dimethylsiloxane) (PDMS) stamp appears to deliver more defined protein patterns on surfaces, while maintaining bioactivity for a wide range of proteins. Here we have quantitatively compared the distribution of fluorescently labeled proteins deposited using direct pipetting, pin printing and μCP printing with flat stamps onto various functionalized glass surfaces of different contact angles through fluorescent microscopy. The uniformity of the deposited protein spots across deposition techniques was also qualitatively analyzed. It was found that with the use of either the direct pipetting or pin printing techniques that protein concentration on surfaces varied largely across surfaces with different contact angles, whereas adsorption did not vary significantly when using the μCP printing Furthermore, when μCP printing was performed with flat relief structures the spot inhomogeneity was lower than when classical methods were used, and even less so when a pyramid relief structure was used. This suggests that μCP printing with pyramid relief structures could produce protein patterns on various surfaces and with increased spot uniformity to enable more reliable protein microarrays.

  15. Geometry sensing by self-organized protein patterns.

    PubMed

    Schweizer, Jakob; Loose, Martin; Bonny, Mike; Kruse, Karsten; Mönch, Ingolf; Schwille, Petra

    2012-09-18

    In the living cell, proteins are able to organize space much larger than their dimensions. In return, changes of intracellular space can influence biochemical reactions, allowing cells to sense their size and shape. Despite the possibility to reconstitute protein self-organization with only a few purified components, we still lack knowledge of how geometrical boundaries affect spatiotemporal protein patterns. Following a minimal systems approach, we used purified proteins and photolithographically patterned membranes to study the influence of spatial confinement on the self-organization of the Min system, a spatial regulator of bacterial cytokinesis, in vitro. We found that the emerging protein pattern responds even to the lateral, two-dimensional geometry of the membrane such that, as in the three-dimensional cell, Min protein waves travel along the longest axis of the membrane patch. This shows that for spatial sensing the Min system does not need to be enclosed in a three-dimensional compartment. Using a computational model we quantitatively analyzed our experimental findings and identified persistent binding of MinE to the membrane as requirement for the Min system to sense geometry. Our results give insight into the interplay between geometrical confinement and biochemical patterns emerging from a nonlinear reaction-diffusion system.

  16. Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood.

    PubMed

    Park, Yang-Seok; Sunkara, Vijaya; Kim, Yubin; Lee, Won Seok; Han, Ja-Ryoung; Cho, Yoon-Kyoung

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins. PMID:27167836

  17. Coupling electrodeposition with layer-by-layer assembly to address proteins within microfluidic channels.

    PubMed

    Wang, Yifeng; Liu, Yi; Cheng, Yi; Kim, Eunkyoung; Rubloff, Gary W; Bentley, William E; Payne, Gregory F

    2011-12-22

    Two thin-film assembly methods are coupled to address proteins. Electrodeposition confers programmability and generates a template for layer-by-layer (LbL) assembly. LbL enables precise control of film thickness and the incorporation of labile biological components. The capabilities are demonstrated using glucose oxidase (GOx) based electrochemical biosensing within a microfabricated fluidic device.

  18. A Microfluidic Bioreactor with in Situ SERS Imaging for the Study of Controlled Flow Patterns of Biofilm Precursor Materials

    PubMed Central

    Paquet-Mercier, François; Aznaveh, Nahid Babaei; Safdar, Muhammad; Greener, Jesse

    2013-01-01

    A microfluidic bioreactor with an easy to fabricate nano-plasmonic surface is demonstrated for studies of biofilms and their precursor materials via Surface Enhanced Raman Spectroscopy (SERS). The system uses a novel design to induce sheath flow confinement of a sodium citrate biofilm precursor stream against the SERS imaging surface to measure spatial variations in the concentration profile. The unoptimised SERS enhancement was approximately 2.5 × 104, thereby improving data acquisition time, reducing laser power requirements and enabling a citrate detection limit of 0.1 mM, which was well below the concentrations used in biofilm nutrient solutions. The flow confinement was observed by both optical microscopy and SERS imaging with good complementarity. We demonstrate the new bioreactor by growing flow-templated biofilms on the microchannel wall. This work opens the way for in situ spectral imaging of biofilms and their biochemical environment under dynamic flow conditions. PMID:24172286

  19. Turn prediction in proteins using a pattern-matching approach.

    PubMed

    Cohen, F E; Abarbanel, R M; Kuntz, I D; Fletterick, R J

    1986-01-14

    We extend the use of amino acid sequence patterns [Cohen, F.E., Abarbanel, R. M., Kuntz, I. D., & Fletterick, R. J. (1983) Biochemistry 22, 4894-4904] to the identification of turns in globular proteins. The approach uses a conservative strategy, combined with a hierarchical search (strongest patterns first) and length-dependent masking, to achieve high accuracy (95%) on a test set of proteins of known structure. Applying the same procedure to homologous families gives a 90% success rate. Straightforward changes are suggested to improve the predictive power. The computer program, written in Lisp, provides a general pattern-recognition language well suited for a number of investigations of protein and nucleic acid sequences. PMID:3754149

  20. Microfluidic technologies.

    PubMed

    Bhagat, Ali Asgar S; Lim, Chwee Teck

    2012-01-01

    Presence of circulating tumor cells (CTCs) in blood is an important intermediate step in cancer metastasis, a mortal consequence of cancer. However, CTCs are extremely rare in blood with highly heterogeneous morphologies and molecular signatures, thus making their isolation technically very challenging. In the past decade, a flurry of new microfluidic-based technologies has emerged to address this compelling problem. This chapter highlights the current state of the art in microfluidic systems developed for CTCs separation and isolation. The techniques presented are broadly classified as physical- or affinity-based isolation depending on the separation principle. The performance of these techniques is evaluated based on accepted separation metrics including sensitivity, purity and processing/analysis time. Finally, further insights associated with realizing an integrated microfluidic CTC lab-on-chip system as an onco-diagnostic tool will be discussed. PMID:22527494

  1. Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms.

    PubMed

    Otvos, Reka A; Iyer, Janaki Krishnamoorthy; van Elk, René; Ulens, Chris; Niessen, Wilfried M A; Somsen, Govert W; Kini, R Manjunatha; Smit, August B; Kool, Jeroen

    2015-07-01

    The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP) was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS) system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach. PMID:26114334

  2. Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms

    PubMed Central

    Otvos, Reka A.; Krishnamoorthy Iyer, Janaki; van Elk, René; Ulens, Chris; Niessen, Wilfried M. A.; Somsen, Govert W.; Kini, R. Manjunatha; Smit, August B.; Kool, Jeroen

    2015-01-01

    The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP) was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS) system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach. PMID:26114334

  3. Integrated microfluidic platforms for investigating neuronal networks

    NASA Astrophysics Data System (ADS)

    Kim, Hyung Joon

    This dissertation describes the development and application of integrated microfluidics-based assay platforms to study neuronal activities in the nervous system in-vitro. The assay platforms were fabricated using soft lithography and micro/nano fabrication including microfluidics, surface patterning, and nanomaterial synthesis. The use of integrated microfluidics-based assay platform allows culturing and manipulating many types of neuronal tissues in precisely controlled microenvironment. Furthermore, they provide organized multi-cellular in-vitro model, long-term monitoring with live cell imaging, and compatibility with molecular biology techniques and electrophysiology experiment. In this dissertation, the integrated microfluidics-based assay platforms are developed for investigation of neuronal activities such as local protein synthesis, impairment of axonal transport by chemical/physical variants, growth cone path finding under chemical/physical cues, and synaptic transmission in neuronal circuit. Chapter 1 describes the motivation, objectives, and scope for developing in-vitro platform to study various neuronal activities. Chapter 2 introduces microfluidic culture platform for biochemical assay with large-scale neuronal tissues that are utilized as model system in neuroscience research. Chapter 3 focuses on the investigation of impaired axonal transport by beta-Amyloid and oxidative stress. The platform allows to control neuronal processes and to quantify mitochondrial movement in various regions of axons away from applied drugs. Chapter 4 demonstrates the development of microfluidics-based growth cone turning assay to elucidate the mechanism underlying axon guidance under soluble factors and shear flow. Using this platform, the behaviors of growth cone of mammalian neurons are verified under the gradient of inhibitory molecules and also shear flow in well-controlled manner. In Chapter 5, I combine in-vitro multicellular model with microfabricated MEA

  4. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-06-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity.

  5. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed Central

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-01-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity. Images PMID:6286971

  6. Pattern recognition methods for protein functional site prediction.

    PubMed

    Yang, Zheng Rong; Wang, Lipo; Young, Natasha; Trudgian, Dave; Chou, Kuo-Chen

    2005-10-01

    Protein functional site prediction is closely related to drug design, hence to public health. In order to save the cost and the time spent on identifying the functional sites in sequenced proteins in biology laboratory, computer programs have been widely used for decades. Many of them are implemented using the state-of-the-art pattern recognition algorithms, including decision trees, neural networks and support vector machines. Although the success of this effort has been obvious, advanced and new algorithms are still under development for addressing some difficult issues. This review will go through the major stages in developing pattern recognition algorithms for protein functional site prediction and outline the future research directions in this important area. PMID:16248799

  7. Effects of shear on P-selectin deposition in microfluidic channels.

    PubMed

    Shimp, Eddie A; Alsmadi, Nesreen Z; Cheng, Tiffany; Lam, Kevin H; Lewis, Christopher S; Schmidtke, David W

    2016-03-01

    Traditional leukocyte adhesion assays have provided significant insight into the mechanisms of leukocyte rolling in part through the use of homogeneously coated surfaces. These assays typically involve protein coating of glass coverslips or plastic petri dishes applied via a static drop of protein solution. With this approach, it is difficult to spatially control the location of proteins to fabricate surface-bound protein gradients that mimic in vivo situations. Microfluidic patterning of proteins with microfluidic devices has become a popular technique due to the ability to spatially pattern proteins on a cellular scale. Despite the advantages of microfluidic patterning, few studies have systematically investigated the effects of perfusion time, protein concentration, and perfusion shear stress on protein deposition. Herein, we demonstrated the fabrication of both line and step gradients of P-selectin on glass substrates that support cell rolling and adhesion assays. Investigation of the flow conditions during the microfluidic patterning led to several significant findings. We observed that the protein deposition time of 5 min was sufficient to deposit adequate P-selectin to support neutrophil rolling. We demonstrated that the amount of membrane P-selectin (mP-selectin) or recombinant P-selectin (rP-selectin) deposited showed a dependence on the perfusion shear stress between 4.0 and 32.0 dyn/cm(2), while similar studies with fibronectin or fibrinogen showed no shear stress dependence. Finally, we also created step changes in surface adherent protein concentration of P-selectin to characterize leukocyte-rolling behavior in response to sudden changes in ligand density. PMID:27190563

  8. The Microfluidic Jukebox

    PubMed Central

    Tan, Say Hwa; Maes, Florine; Semin, Benoît; Vrignon, Jérémy; Baret, Jean-Christophe

    2014-01-01

    Music is a form of art interweaving people of all walks of life. Through subtle changes in frequencies, a succession of musical notes forms a melody which is capable of mesmerizing the minds of people. With the advances in technology, we are now able to generate music electronically without relying solely on physical instruments. Here, we demonstrate a musical interpretation of droplet-based microfluidics as a form of novel electronic musical instruments. Using the interplay of electric field and hydrodynamics in microfluidic devices, well controlled frequency patterns corresponding to musical tracks are generated in real time. This high-speed modulation of droplet frequency (and therefore of droplet sizes) may also provide solutions that reconciles high-throughput droplet production and the control of individual droplet at production which is needed for many biochemical or material synthesis applications. PMID:24781785

  9. The Microfluidic Jukebox

    NASA Astrophysics Data System (ADS)

    Tan, Say Hwa; Maes, Florine; Semin, Benoît; Vrignon, Jérémy; Baret, Jean-Christophe

    2014-04-01

    Music is a form of art interweaving people of all walks of life. Through subtle changes in frequencies, a succession of musical notes forms a melody which is capable of mesmerizing the minds of people. With the advances in technology, we are now able to generate music electronically without relying solely on physical instruments. Here, we demonstrate a musical interpretation of droplet-based microfluidics as a form of novel electronic musical instruments. Using the interplay of electric field and hydrodynamics in microfluidic devices, well controlled frequency patterns corresponding to musical tracks are generated in real time. This high-speed modulation of droplet frequency (and therefore of droplet sizes) may also provide solutions that reconciles high-throughput droplet production and the control of individual droplet at production which is needed for many biochemical or material synthesis applications.

  10. Solid-phase Extraction and Purification of Membrane Proteins Using a UV-modified PMMA Microfluidic Bioaffinity μSPE Device

    PubMed Central

    Battle, Katrina N.; Jackson, Joshua M.; Witek, Małgorzata A.; Hupert, Mateusz L.; Hunsucker, Sally A.; Armistead, Paul M.; Soper, Steven A.

    2014-01-01

    We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3,600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (~20 fmol) of membrane proteins could be isolated and recovered with ~89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs. PMID:24487280

  11. Protein pattern of Xenopus laevis embryos grown in simulated microgravity.

    PubMed

    Tedeschi, Gabriella; Pagliato, Lara; Negroni, Manuela; Montorfano, Gigliola; Corsetto, Paola; Nonnis, Simona; Negri, Armando; Rizzo, Angela Maria

    2011-03-01

    Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.

  12. Microfluidic Mixing Technology for a Universal Health Sensor

    NASA Technical Reports Server (NTRS)

    Chan, Eugene Y.; Bae, Candice

    2009-01-01

    A highly efficient means of microfluidic mixing has been created for use with the rHEALTH sensor an elliptical mixer and passive curvilinear mixing patterns. The rHEALTH sensor provides rapid, handheld, complete blood count, cell differential counts, electrolyte measurements, and other lab tests based on a reusable, flow-based microfluidic platform. These geometries allow for cleaning in a reusable manner, and also allow for complete mixing of fluid streams. The microfluidic mixing is performed by flowing two streams of fluid into an elliptical or curvilinear design that allows the combination of the flows into one channel. The mixing is accomplished by either chaotic advection around micro - fluidic loops. All components of the microfluidic chip are flow-through, meaning that cleaning solution can be introduced into the chip to flush out cells, plasma proteins, and dye. Tests were performed on multiple chip geometries to show that cleaning is efficient in any flowthrough design. The conclusion from these experiments is that the chip can indeed be flushed out with microliter volumes of solution and biological samples are cleaned readily from the chip with minimal effort. The technology can be applied in real-time health monitoring at patient s bedside or in a doctor s office, and real-time clinical intervention in acute situations. It also can be used for daily measurement of hematocrit for patients on anticoagulant drugs, or to detect acute myocardial damage outside a hospital.

  13. Microfluidic Device

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Lin, Jeffrey Chun-Hui (Inventor); Kasdan, Harvey (Inventor)

    2015-01-01

    Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

  14. Microfluidic Device

    NASA Technical Reports Server (NTRS)

    Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Lin, Jeffrey Chun-Hui (Inventor); Kasdan, Harvey L. (Inventor)

    2016-01-01

    Described herein are particular embodiments relating to a microfluidic device that may be utilized for cell sensing, counting, and/or sorting. Particular aspects relate to a microfabricated device that is capable of differentiating single cell types from dense cell populations. One particular embodiment relates a device and methods of using the same for sensing, counting, and/or sorting leukocytes from whole, undiluted blood samples.

  15. Humidified microcontact printing of proteins: universal patterning of proteins on both low and high energy surfaces.

    PubMed

    Ricoult, Sébastien G; Nezhad, Amir Sanati; Knapp-Mohammady, Michaela; Kennedy, Timothy E; Juncker, David

    2014-10-14

    Microcontact printing (μCP) of proteins is widely used for biosensors and cell biology but is constrained to printing proteins adsorbed to a low free energy, hydrophobic surface to a high free energy, hydrophilic surface. This strongly limits μCP as harsh chemical treatments are required to form a high energy surface. Here, we introduce humidified μCP (HμCP) of proteins which enables universal printing of protein on any smooth surface. We found that by flowing water in proximity to proteins adsorbed on a hydrophilized stamp, the water vapor diffusing through the stamp enables the printing of proteins on both low and high energy surfaces. Indeed, when proteins are printed using stamps with increasing spacing between water-filled microchannels, only proteins adjacent to the channels are transferred. The vapor transport through the stamp was modeled, and by comparing the humidity profiles with the protein patterns, 88% relative humidity in the stamp was identified as the threshold for HμCP. The molecular forces occurring between PDMS, peptides, and glass during printing were modeled ab initio to confirm the critical role water plays in the transfer. Using HμCP, we introduce straightforward protocols to pattern multiple proteins side-by-side down to nanometer resolution without the need for expensive mask aligners, but instead exploiting self-alignment effects derived from the stamp geometry. Finally, we introduce vascularized HμCP stamps with embedded microchannels that allow printing proteins as arbitrary, large areas patterns with nanometer resolution. This work introduces the general concept of water-assisted μCP and opens new possibilities for "solvent-assisted" printing of proteins and of other nanoparticles. PMID:25222734

  16. Humidified microcontact printing of proteins: universal patterning of proteins on both low and high energy surfaces.

    PubMed

    Ricoult, Sébastien G; Nezhad, Amir Sanati; Knapp-Mohammady, Michaela; Kennedy, Timothy E; Juncker, David

    2014-10-14

    Microcontact printing (μCP) of proteins is widely used for biosensors and cell biology but is constrained to printing proteins adsorbed to a low free energy, hydrophobic surface to a high free energy, hydrophilic surface. This strongly limits μCP as harsh chemical treatments are required to form a high energy surface. Here, we introduce humidified μCP (HμCP) of proteins which enables universal printing of protein on any smooth surface. We found that by flowing water in proximity to proteins adsorbed on a hydrophilized stamp, the water vapor diffusing through the stamp enables the printing of proteins on both low and high energy surfaces. Indeed, when proteins are printed using stamps with increasing spacing between water-filled microchannels, only proteins adjacent to the channels are transferred. The vapor transport through the stamp was modeled, and by comparing the humidity profiles with the protein patterns, 88% relative humidity in the stamp was identified as the threshold for HμCP. The molecular forces occurring between PDMS, peptides, and glass during printing were modeled ab initio to confirm the critical role water plays in the transfer. Using HμCP, we introduce straightforward protocols to pattern multiple proteins side-by-side down to nanometer resolution without the need for expensive mask aligners, but instead exploiting self-alignment effects derived from the stamp geometry. Finally, we introduce vascularized HμCP stamps with embedded microchannels that allow printing proteins as arbitrary, large areas patterns with nanometer resolution. This work introduces the general concept of water-assisted μCP and opens new possibilities for "solvent-assisted" printing of proteins and of other nanoparticles.

  17. Assembly of designed protein scaffolds into monolayers for nanoparticle patterning.

    PubMed

    Mejias, Sara H; Couleaud, Pierre; Casado, Santiago; Granados, Daniel; Garcia, Miguel Angel; Abad, Jose M; Cortajarena, Aitziber L

    2016-05-01

    The controlled assembly of building blocks to achieve new nanostructured materials with defined properties at different length scales through rational design is the basis and future of bottom-up nanofabrication. This work describes the assembly of the idealized protein building block, the consensus tetratricopeptide repeat (CTPR), into monolayers by oriented immobilization of the blocks. The selectivity of thiol-gold interaction for an oriented immobilization has been verified by comparing a non-thiolated protein building block. The physical properties of the CTPR protein thin biomolecular films including topography, thickness, and viscoelasticity, are characterized. Finally, the ability of these scaffolds to act as templates for inorganic nanostructures has been demonstrated by the formation of well-packed gold nanoparticles (GNPs) monolayer patterned by the CTPR monolayer.

  18. Functional module identification in protein interaction networks by interaction patterns

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

    Motivation: Identifying functional modules in protein–protein interaction (PPI) networks may shed light on cellular functional organization and thereafter underlying cellular mechanisms. Many existing module identification algorithms aim to detect densely connected groups of proteins as potential modules. However, based on this simple topological criterion of ‘higher than expected connectivity’, those algorithms may miss biologically meaningful modules of functional significance, in which proteins have similar interaction patterns to other proteins in networks but may not be densely connected to each other. A few blockmodel module identification algorithms have been proposed to address the problem but the lack of global optimum guarantee and the prohibitive computational complexity have been the bottleneck of their applications in real-world large-scale PPI networks. Results: In this article, we propose a novel optimization formulation LCP2 (low two-hop conductance sets) using the concept of Markov random walk on graphs, which enables simultaneous identification of both dense and sparse modules based on protein interaction patterns in given networks through searching for LCP2 by random walk. A spectral approximate algorithm SLCP2 is derived to identify non-overlapping functional modules. Based on a bottom-up greedy strategy, we further extend LCP2 to a new algorithm (greedy algorithm for LCP2) GLCP2 to identify overlapping functional modules. We compare SLCP2 and GLCP2 with a range of state-of-the-art algorithms on synthetic networks and real-world PPI networks. The performance evaluation based on several criteria with respect to protein complex prediction, high level Gene Ontology term prediction and especially sparse module detection, has demonstrated that our algorithms based on searching for LCP2 outperform all other compared algorithms. Availability and implementation: All data and code are available at http://www.cse.usf.edu/∼xqian/fmi/slcp2hop

  19. Microfluidic interconnects

    DOEpatents

    Benett, William J.; Krulevitch, Peter A.

    2001-01-01

    A miniature connector for introducing microliter quantities of solutions into microfabricated fluidic devices. The fluidic connector, for example, joins standard high pressure liquid chromatography (HPLC) tubing to 1 mm diameter holes in silicon or glass, enabling ml-sized volumes of sample solutions to be merged with .mu.l-sized devices. The connector has many features, including ease of connect and disconnect; a small footprint which enables numerous connectors to be located in a small area; low dead volume; helium leak-tight; and tubing does not twist during connection. Thus the connector enables easy and effective change of microfluidic devices and introduction of different solutions in the devices.

  20. Microfluidic device having an immobilized pH gradient and page gels for protein separation and analysis

    SciTech Connect

    Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

    2014-05-20

    Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

  1. Microfluidic device having an immobilized pH gradient and PAGE gels for protein separation and analysis

    DOEpatents

    Sommer, Gregory J.; Hatch, Anson V.; Singh, Anup K.; Wang, Ying-Chih

    2012-12-11

    Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

  2. Tacky COC: a solvent bonding technique for fabrication of microfluidic systems

    NASA Astrophysics Data System (ADS)

    Keller, Nico; Nargang, Tobias M.; Helmer, Dorothea; Rapp, Bastian E.

    2016-03-01

    The academic community knows cyclic olefin copolymer (COC) as a well suited material for microfluidic applications because COC has numerous interesting properties such as high transmittance, good chemical resistance and good biocompatibility. Here we present a fast and cost-effective method for bonding of two COC substrates: exposure to appropriate solvents gives a tacky COC surface which when brought in contact with untreated COC forms a strong and optical clear bond. The bonding process is carried out at room temperature and takes less than three minutes which makes it significantly faster than currently described methods: This method does not require special lab equipment such as hot plates or hydraulic presses. The mild conditions of the bond process also allow for such "tacky COC" lids to be used for sealing of microfluidic chips containing immobilized protein patterns which is of high interest for immunodiagnostic testing inside microfluidic chips.

  3. Pattern Formation by Electrostatic Self-Organization of Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Boedec, G.; Jaeger, M.; Homble, F.; Leonetti, M.

    2012-07-01

    The electric activity of biological cells and organs such as heart for example is at the origin of various phenomena of pattern formation. The electric membrane potential appears as the order parameter to characterize these spatiotemporal dynamics. A kind of patterns is characterized by a stationary spatial modulation of membrane potential along the cell, breaking a symmetry of the system. They are associated to transcellular currents. A mechanism proposed in literature is based on the coupling of the electric current produced by membrane proteins and their electrophoretic mobilities. Beyond its classical linear stability analysis, the numerical and theoretical analysis of this model offers a variety of spatiotemporal dynamics. Firstly, the background in the modelization of electric phenomena is recalled. Secondly, the analysis is focused on two nonlinear dynamics.

  4. Pbx Homeodomain Proteins: TALEnted regulators of Limb Patterning and Outgrowth

    PubMed Central

    Capellini, Terence D.; Zappavigna, Vincenzo; Selleri, Licia

    2011-01-01

    Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5' HoxA/D gene expression. PMID:21416555

  5. Laser Ablation of Polymer Microfluidic Devices

    NASA Astrophysics Data System (ADS)

    Killeen, Kevin

    2004-03-01

    Microfluidic technology is ideal for processing precious samples of limited volumes. Some of the most important classes of biological samples are both high in sample complexity and low in concentration. Combining the elements of sample pre-concentration, chemical separation and high sensitivity detection with chemical identification is essential for realizing a functional microfluidic based analysis system. Direct write UV laser ablation has been used to rapidly fabricate microfluidic devices capable of high performance liquid chromatography (HPLC)-MS. These chip-LC/MS devices use bio-compatible, solvent resistant and flexible polymer materials such as polyimide. A novel microfluidic to rotary valve interface enables, leak free, high pressure fluid switching between multiple ports of the microfluidic chip-LC/MS device. Electrospray tips with outer dimension of 50 um and inner of 15 um are formed by ablating the polymer material concentrically around a multilayer laminated channel structure. Biological samples of digested proteins were used to evaluate the performance of these microfluidic devices. Liquid chromatography separation and similar sample pretreatments have been performed using polymeric microfluidic devices with on-chip separation channels. Mass spectrometry was performed using an Agilent Technologies 1100 series ion trap mass spectrometer. Low fmol amounts of protein samples were positively and routinely identified by searching the MS/MS spectral data against protein databases. The sensitivity and separation performance of the chip-LC devices has been found to be comparable to state of the art nano-electrospray systems.

  6. Using macromolecular-crystallography beamline and microfluidic platform for small-angle diffraction studies of lipidic matrices for membrane-protein crystallization

    PubMed Central

    Kondrashkina, E; Khvostichenko, D S; Perry, S L; Von Osinski, J; Kenis, P J A; Brister, K

    2013-01-01

    Macromolecular-crystallography (MX) beamlines routinely provide a possibility to change X-ray beam energy, focus the beam to a size of tens of microns, align a sample on a microdiffractometer using on-axis video microscope, and collect data with an area-detector positioned in three dimensions. These capabilities allow for running complementary measurements of small-angle X-ray scattering and diffraction (SAXS) at the same beamline with such additions to the standard MX setup as a vacuum path between the sample and the detector, a modified beam stop, and a custom sample cell. On the 21-ID-D MX beamline at the Advanced Photon Source we attach a vacuum flight tube to the area detector support and use the support motion for aligning a beam stop built into the rear end of the flight tube. At 8 KeV energy and 1 m sample-to-detector distance we can achieve a small-angle resolution of 0.01A−1 in the reciprocal space. Measuring SAXS with this setup, we have studied phase diagrams of lipidic mesophases used as matrices for membrane-protein crystallization. The outcome of crystallization trials is significantly affected by the structure of the lipidic mesophases, which is determined by the composition of the crystallization mixture. We use a microfluidic chip for the mesophase formulation and in situ SAXS data collection. Using the MX beamline and the microfluidic platform we have demonstrated the viability of the high-throughput SAXS studies facilitating screening of lipidic matrices for membrane-protein crystallization. PMID:24260038

  7. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  8. Morphology and protein patterns of honey bee drone accessory glands.

    PubMed

    Cruz-Landim, Carminda da; Dallacqua, Rodrigo Pires

    2005-09-30

    We used light and transmission electron microscopy to examine the morphology of the accessory glands of immature and mature adult males of Apis mellifera L. We also made an electrophoretic analysis of the protein content of the mature gland. The glands of the immature male actively secrete a mucous substance that can be seen in the lumen of the gland of the mature male. This secretion stains with mercury bromophenol blue and with periodic acid-Schiff reaction, which stain glyconjugates. The protein content was higher in the lumen secretion than in the gland wall extracts. The electrophoresis patterns of the wall extracts were different from those of the secretion found in the gland lumen.

  9. Morphology and protein patterns of honey bee drone accessory glands.

    PubMed

    Cruz-Landim, Carminda da; Dallacqua, Rodrigo Pires

    2005-01-01

    We used light and transmission electron microscopy to examine the morphology of the accessory glands of immature and mature adult males of Apis mellifera L. We also made an electrophoretic analysis of the protein content of the mature gland. The glands of the immature male actively secrete a mucous substance that can be seen in the lumen of the gland of the mature male. This secretion stains with mercury bromophenol blue and with periodic acid-Schiff reaction, which stain glyconjugates. The protein content was higher in the lumen secretion than in the gland wall extracts. The electrophoresis patterns of the wall extracts were different from those of the secretion found in the gland lumen. PMID:16342031

  10. Droplet microfluidics.

    PubMed

    Teh, Shia-Yen; Lin, Robert; Hung, Lung-Hsin; Lee, Abraham P

    2008-02-01

    Droplet-based microfluidic systems have been shown to be compatible with many chemical and biological reagents and capable of performing a variety of "digital fluidic" operations that can be rendered programmable and reconfigurable. This platform has dimensional scaling benefits that have enabled controlled and rapid mixing of fluids in the droplet reactors, resulting in decreased reaction times. This, coupled with the precise generation and repeatability of droplet operations, has made the droplet-based microfluidic system a potent high throughput platform for biomedical research and applications. In addition to being used as microreactors ranging from the nano- to femtoliter range; droplet-based systems have also been used to directly synthesize particles and encapsulate many biological entities for biomedicine and biotechnology applications. This review will focus on the various droplet operations, as well as the numerous applications of the system. Due to advantages unique to droplet-based systems, this technology has the potential to provide novel solutions to today's biomedical engineering challenges for advanced diagnostics and therapeutics.

  11. Microsecond protein folding events revealed by time-resolved fluorescence resonance energy transfer in a microfluidic mixer.

    PubMed

    Jiang, Liguo; Zeng, Yan; Sun, Qiqi; Sun, Yueru; Guo, Zhihong; Qu, Jianan Y; Yao, Shuhuai

    2015-06-01

    We demonstrate the combination of the time-resolved fluorescence resonance energy transfer (tr-FRET) measurement and the ultrarapid hydrodynamic focusing microfluidic mixer. The combined technique is capable of probing the intermolecular distance change with temporal resolution at microsecond level and structural resolution at Angstrom level, and the use of two-photon excitation enables a broader exploration of FRET with spectrum from near-ultraviolet to visible wavelength. As a proof of principle, we used the coupled microfluidic laminar flow and time-resolved two-photon excitation microscopy to investigate the early folding states of Cytochrome c (cyt c) by monitoring the distance between the tryptophan (Trp-59)-heme donor-acceptor (D-A) pair. The transformation of folding states of cyt c in the early 500 μs of refolding was revealed on the microsecond time scale. For the first time, we clearly resolved the early transient state of cyt c, which is populated within the dead time of the mixer (<10 μs) and has a characteristic Trp-59-heme distance of ∼31 Å. We believe this tool can find more applications in studying the early stages of biological processes with FRET as the probe.

  12. Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

    PubMed Central

    Batoulis, Helena; Schmidt, Thomas H.; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

    2016-01-01

    Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems. PMID:27052788

  13. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    }E4, these kinases regulate one of the E1{sup ∧}E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4. - Highlights: • E4 gene products have a modular structure, and are expressed from the E1{sup ∧}E4 spliced mRNA. • E4 proteins are modified during epithelial differentiation by phosphorylation and proteolysis. • The E4 proteins contribute to genome amplification-efficiency and virus synthesis. • E4 proteins are abundantly expressed and may facilitate efficient virus release and transmission. • High-risk E4 proteins are deposited as amyloid fibres and can be used as infection biomarkers.

  14. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    SciTech Connect

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.

  15. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    DOE PAGES

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR formore » nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.« less

  16. Semiconductor sensor embedded microfluidic chip for protein biomarker detection using a bead-based immunoassay combined with deoxyribonucleic acid strand labeling.

    PubMed

    Lin, Yen-Heng; Peng, Po-Yu

    2015-04-15

    Two major issues need to be addressed in applying semiconductor biosensors to detecting proteins in immunoassays. First, the length of the antibody on the sensor surface surpasses the Debye lengths (approximately 1 nm, in normal ionic strength solution), preventing certain specifically bound proteins from being tightly attached to the sensor surface. Therefore, these proteins do not contribute to the sensor's surface potential change. Second, these proteins carry a small charge and can be easily affected by the pH of the surrounding solution. This study proposes a magnetic bead-based immunoassay using a secondary antibody to label negatively charged DNA fragments for signal amplification. An externally imposed magnetic force attaches the analyte tightly to the sensor surface, thereby effectively solving the problem of the analyte protein's distance to the sensor surface surpassing the Debye lengths. In addition, a normal ion intensity buffer can be used without dilution for the proposed method. Experiments revealed that the sensitivity can be improved by using a longer DNA fragment for labeling and smaller magnetic beads as solid support for the antibody. By using a 90 base pair DNA label, the signal was 15 times greater than that without labeling. In addition, by using a 120 nm magnetic bead, a minimum detection limit of 12.5 ng mL(-1) apolipoprotein A1 can be measured. Furthermore, this study integrates a semiconductor sensor with a microfluidic chip. With the help of microvalves and micromixers in the chip, the length of the mixing step for each immunoassay has been reduced from 1h to 20 min, and the sample volume has been reduced from 80 μL to 10 μL. In practice, a protein biomarker in a urinary bladder cancer patient's urine was successfully measured using this technique. This study provides a convenient and effective method to measure protein using a semiconductor sensor.

  17. Microfluidic electrochemical reactors

    DOEpatents

    Nuzzo, Ralph G.; Mitrovski, Svetlana M.

    2011-03-22

    A microfluidic electrochemical reactor includes an electrode and one or more microfluidic channels on the electrode, where the microfluidic channels are covered with a membrane containing a gas permeable polymer. The distance between the electrode and the membrane is less than 500 micrometers. The microfluidic electrochemical reactor can provide for increased reaction rates in electrochemical reactions using a gaseous reactant, as compared to conventional electrochemical cells. Microfluidic electrochemical reactors can be incorporated into devices for applications such as fuel cells, electrochemical analysis, microfluidic actuation, pH gradient formation.

  18. Microfluidic interconnects

    DOEpatents

    Benett, William J.; Krulevitch, Peter A.

    2001-01-01

    A miniature connector for introducing microliter quantities of solutions into microfabricated fluidic devices, and which incorporates a molded ring or seal set into a ferrule cartridge, with or without a compression screw. The fluidic connector, for example, joins standard high pressure liquid chromatography (HPLC) tubing to 1 mm diameter holes in silicon or glass, enabling ml-sized volumes of sample solutions to be merged with .mu.l-sized devices. The connector has many features, including ease of connect and disconnect; a small footprint which enables numerous connectors to be located in a small area; low dead volume; helium leak-tight; and tubing does not twist during connection. Thus the connector enables easy and effective change of microfluidic devices and introduction of different solutions in the devices.

  19. Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

    PubMed Central

    Hou, Yanxia; Genua, Maria; Garçon, Laurie-Amandine; Buhot, Arnaud; Calemczuk, Roberto; Bonnaffé, David; Lortat-Jacob, Hugues; Livache, Thierry

    2014-01-01

    In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins. PMID:25286325

  20. Laterally Mobile, Functionalized Self-Assembled Monolayers at the Fluorous−Aqueous Interface in a Plug-Based Microfluidic System: Characterization and Testing with Membrane Protein Crystallization

    SciTech Connect

    Kreutz, Jason E.; Li, Liang; Roach, L. Spencer; Hatakeyama, Takuji; Ismagilov, Rustem F.

    2009-11-04

    This paper describes a method to generate functionalizable, mobile self-assembled monolayers (SAMs) in plug-based microfluidics. Control of interfaces is advancing studies of biological interfaces, heterogeneous reactions, and nanotechnology. SAMs have been useful for such studies, but they are not laterally mobile. Lipid-based methods, though mobile, are not easily amenable to setting up the hundreds of experiments necessary for crystallization screening. Here we demonstrate a method, complementary to current SAM and lipid methods, for rapidly generating mobile, functionalized SAMs. This method relies on plugs, droplets surrounded by a fluorous carrier fluid, to rapidly explore chemical space. Specifically, we implemented his-tag binding chemistry to design a new fluorinated amphiphile, RfNTA, using an improved one-step synthesis of RfOEG under Mitsunobu conditions. RfNTA introduces specific binding of protein at the fluorous-aqueous interface, which concentrates and orients proteins at the interface, even in the presence of other surfactants. We then applied this approach to the crystallization of a his-tagged membrane protein, Reaction Center from Rhodobacter sphaeroides, performed 2400 crystallization trials, and showed that this approach can increase the range of crystal-producing conditions, the success rate at a given condition, the rate of nucleation, and the quality of the crystal formed.

  1. Reactions in droplets in microfluidic channels.

    PubMed

    Song, Helen; Chen, Delai L; Ismagilov, Rustem F

    2006-11-13

    Fundamental and applied research in chemistry and biology benefits from opportunities provided by droplet-based microfluidic systems. These systems enable the miniaturization of reactions by compartmentalizing reactions in droplets of femoliter to microliter volumes. Compartmentalization in droplets provides rapid mixing of reagents, control of the timing of reactions on timescales from milliseconds to months, control of interfacial properties, and the ability to synthesize and transport solid reagents and products. Droplet-based microfluidics can help to enhance and accelerate chemical and biochemical screening, protein crystallization, enzymatic kinetics, and assays. Moreover, the control provided by droplets in microfluidic devices can lead to new scientific methods and insights.

  2. Inertial microfluidic physics.

    PubMed

    Amini, Hamed; Lee, Wonhee; Di Carlo, Dino

    2014-08-01

    Microfluidics has experienced massive growth in the past two decades, and especially with advances in rapid prototyping researchers have explored a multitude of channel structures, fluid and particle mixtures, and integration with electrical and optical systems towards solving problems in healthcare, biological and chemical analysis, materials synthesis, and other emerging areas that can benefit from the scale, automation, or the unique physics of these systems. Inertial microfluidics, which relies on the unconventional use of fluid inertia in microfluidic platforms, is one of the emerging fields that make use of unique physical phenomena that are accessible in microscale patterned channels. Channel shapes that focus, concentrate, order, separate, transfer, and mix particles and fluids have been demonstrated, however physical underpinnings guiding these channel designs have been limited and much of the development has been based on experimentally-derived intuition. Here we aim to provide a deeper understanding of mechanisms and underlying physics in these systems which can lead to more effective and reliable designs with less iteration. To place the inertial effects into context we also discuss related fluid-induced forces present in particulate flows including forces due to non-Newtonian fluids, particle asymmetry, and particle deformability. We then highlight the inverse situation and describe the effect of the suspended particles acting on the fluid in a channel flow. Finally, we discuss the importance of structured channels, i.e. channels with boundary conditions that vary in the streamwise direction, and their potential as a means to achieve unprecedented three-dimensional control over fluid and particles in microchannels. Ultimately, we hope that an improved fundamental and quantitative understanding of inertial fluid dynamic effects can lead to unprecedented capabilities to program fluid and particle flow towards automation of biomedicine, materials

  3. Tacky cyclic olefin copolymer: a biocompatible bonding technique for the fabrication of microfluidic channels in COC.

    PubMed

    Keller, Nico; Nargang, Tobias M; Runck, Matthias; Kotz, Frederik; Striegel, Andreas; Sachsenheimer, Kai; Klemm, Denis; Länge, Kerstin; Worgull, Matthias; Richter, Christiane; Helmer, Dorothea; Rapp, Bastian E

    2016-04-26

    Cyclic olefin copolymer (COC) is widely used in microfluidics due to its UV-transparency, its biocompatibility and high chemical resistance. Here we present a fast and cost-effective solvent bonding technique, which allows for the efficient bonding of protein-patterned COC structures. The bonding process is carried out at room temperature and takes less than three minutes. Enzyme activity is retained upon bonding and microstructure deformation does not occur. PMID:27040493

  4. Microfluidic devices: useful tools for bioprocess intensification.

    PubMed

    Marques, Marco P C; Fernandes, Pedro

    2011-01-01

    The dawn of the new millennium saw a trend towards the dedicated use of microfluidic devices for process intensification in biotechnology. As the last decade went by, it became evident that this pattern was not a short-lived fad, since the deliverables related to this field of research have been consistently piling-up. The application of process intensification in biotechnology is therefore seemingly catching up with the trend already observed in the chemical engineering area, where the use of microfluidic devices has already been upgraded to production scale. The goal of the present work is therefore to provide an updated overview of the developments centered on the use of microfluidic devices for process intensification in biotechnology. Within such scope, particular focus will be given to different designs, configurations and modes of operation of microreactors, but reference to similar features regarding microfluidic devices in downstream processing will not be overlooked. Engineering considerations and fluid dynamics issues, namely related to the characterization of flow in microchannels, promotion of micromixing and predictive tools, will also be addressed, as well as reflection on the analytics required to take full advantage of the possibilities provided by microfluidic devices in process intensification. Strategies developed to ease the implementation of experimental set-ups anchored in the use of microfluidic devices will be briefly tackled. Finally, realistic considerations on the current advantages and limitation on the use of microfluidic devices for process intensification, as well as prospective near future developments in the field, will be presented.

  5. Protein patterning by microcontact printing using pyramidal PDMS stamps.

    PubMed

    Filipponi, Luisa; Livingston, Peter; Kašpar, Ondřej; Tokárová, Viola; Nicolau, Dan V

    2016-02-01

    Micro-contact printing, μCP, is a well-established soft-lithography technique for printing biomolecules. μCP uses stamps made of Poly(dimethylsiloxane), PDMS, made by replicating a microstructured silicon master fabricated by semiconductor manufacturing processes. One of the problems of the μCP is the difficult control of the printing process, which, because of the high compressibility of PDMS, is very sensitive to minute changes in the applied pressure. This over-sensitive response leads to frequent and/or uncontrollable collapse of the stamps with high aspect ratios, thus decreasing the printing accuracy and reproducibility. Here we present a straightforward methodology of designing and fabricating PDMS structures with an architecture which uses the collapse of the stamp to reduce, rather than enlarge the variability of the printing. The PDMS stamp, organized as an array of pyramidal micro-posts, whose ceiling collapses when pressed on a flat surface, replicates the structure of the silicon master fabricated by anisotropic wet etching. Upon application of pressure, depending on the size of, and the pitch between, the PDMS pyramids, an air gap is formed surrounding either the entire array, or individual posts. The printing technology, which also exhibits a remarkably low background noise for fluorescence detection, may find applications when the clear demarcation of the shapes of protein patterns and the distance between them are critical, such as microarrays and studies of cell patterning. PMID:26782964

  6. PREFACE: Nano- and microfluidics Nano- and microfluidics

    NASA Astrophysics Data System (ADS)

    Jacobs, Karin

    2011-05-01

    The field of nano- and microfluidics emerged at the end of the 1990s parallel to the demand for smaller and smaller containers and channels for chemical, biochemical and medical applications such as blood and DNS analysis [1], gene sequencing or proteomics [2, 3]. Since then, new journals and conferences have been launched and meanwhile, about two decades later, a variety of microfluidic applications are on the market. Briefly, 'the small flow becomes mainstream' [4]. Nevertheless, research in nano- and microfluidics is more than downsizing the spatial dimensions. For liquids on the nanoscale, surface and interface phenomena grow in importance and may even dominate the behavior in some systems. The studies collected in this special issue all concentrate on these type of systems and were part ot the priority programme SPP1164 'Nano- and Microfluidics' of the German Science Foundation (Deutsche Forschungsgemeinschaft, DFG). The priority programme was initiated in 2002 by Hendrik Kuhlmann and myself and was launched in 2004. Friction between a moving liquid and a solid wall may, for instance, play an important role so that the usual assumption of a no-slip boundary condition is no longer valid. Likewise, the dynamic deformations of soft objects like polymers, vesicles or capsules in flow arise from the subtle interplay between the (visco-)elasticity of the object and the viscous stresses in the surrounding fluid and, potentially, the presence of structures confining the flow like channels. Consequently, new theories were developed ( see articles in this issue by Münch and Wagner, Falk and Mecke, Bonthuis et al, Finken et al, Almenar and Rauscher, Straube) and experiments were set up to unambiguously demonstrate deviations from bulk, or 'macro', behavior (see articles in this issue by Wolff et al, Vinogradova and Belyaev, Hahn et al, Seemann et al, Grüner and Huber, Müller-Buschbaum et al, Gutsche et al, Braunmüller et al, Laube et al, Brücker, Nottebrock et al

  7. Microfluidic System for Solution Array Based Bioassays

    SciTech Connect

    Dougherty, G M; Tok, J B; Pannu, S S; Rose, K A

    2006-02-10

    The objective of this project is to demonstrate new enabling technology for multiplex biodetection systems that are flexible, miniaturizable, highly automated, low cost, and high performance. It builds on prior successes at LLNL with particle-based solution arrays, such as those used in the Autonomous Pathogen Detection System (APDS) successfully field deployed to multiple locations nationwide. We report the development of a multiplex solution array immunoassay based upon engineered metallic nanorod particles. Nanobarcodes{reg_sign} particles are fabricated by sequential electrodeposition of dissimilar metals within porous alumina templates, yielding optically encoded striping patterns that can be read using standard laboratory microscope optics and PC-based image processing software. The addition of self-assembled monolayer (SAM) coatings and target-specific antibodies allows each encoded class of nanorod particles to be directed against a different antigen target. A prototype assay panel directed against bacterial, viral, and soluble protein targets demonstrates simultaneous detection at sensitivities comparable to state of the art immunoassays, with minimal cross-reactivity. Studies have been performed to characterize the colloidal properties (zeta potential) of the suspended nanorod particles as a function of pH, the ionic strength of the suspending solution, and surface functionalization state. Additional studies have produced means for the non-contact manipulation of the particles, including the insertion of magnetic nickel stripes within the encoding pattern, and control via externally applied electromagnetic fields. Using the results of these studies, the novel Nanobarcodes{reg_sign} based assay was implemented in a prototype automated system with the sample processing functions and optical readout performed on a microfluidic card. The unique physical properties of the nanorod particles enable the development of integrated microfluidic systems for

  8. Microfluidic sieve valves

    DOEpatents

    Quake, Stephen R; Marcus, Joshua S; Hansen, Carl L

    2015-01-13

    Sieve valves for use in microfluidic device are provided. The valves are useful for impeding the flow of particles, such as chromatography beads or cells, in a microfluidic channel while allowing liquid solution to pass through the valve. The valves find particular use in making microfluidic chromatography modules.

  9. Fully integrated PDMS/SU-8/quartz microfluidic chip with a novel macroporous poly dimethylsiloxane (PDMS) membrane for isoelectric focusing of proteins using whole-channel imaging detection.

    PubMed

    Shameli, Seyed Mostafa; Elbuken, Caglar; Ou, Junjie; Ren, Carolyn L; Pawliszyn, Janusz

    2011-02-01

    A fully integrated polydimethylsiloxane (PDMS)/modified PDMS membrane/SU-8/quartz hybrid chip was developed for protein separation using isoelectric focusing (IEF) mechanism coupled with whole-channel imaging detection (WCID) method. This microfluidic chip integrates three components into one single chip: (i) modified PDMS membranes for separating electrolytes in the reservoirs from the sample in the microchannel and thus reducing pressure disturbance, (ii) SU-8 optical slit to block UV light (below 300 nm) outside the channel aiming to increase detection sensitivity, and (iii) injection and discharge capillaries for continuous operation. Integration of all these components on a single chip is challenging because it requires fabrication techniques for perfect bonding between different materials and is prone to leakage and blockage. This study has addressed all the challenges and presented a fully integrated chip, which is more robust with higher sensitivity than the previously developed IEF chips. This chip was tested by performing protein and pI marker separation. The separation results obtained in this chip were compared with that obtained in commercial cartridges. Side-by-side comparison validated the developed chip and fabrication techniques.

  10. Electro-Microfluidic Packaging

    NASA Astrophysics Data System (ADS)

    Benavides, G. L.; Galambos, P. C.

    2002-06-01

    There are many examples of electro-microfluidic products that require cost effective packaging solutions. Industry has responded to a demand for products such as drop ejectors, chemical sensors, and biological sensors. Drop ejectors have consumer applications such as ink jet printing and scientific applications such as patterning self-assembled monolayers or ejecting picoliters of expensive analytes/reagents for chemical analysis. Drop ejectors can be used to perform chemical analysis, combinatorial chemistry, drug manufacture, drug discovery, drug delivery, and DNA sequencing. Chemical and biological micro-sensors can sniff the ambient environment for traces of dangerous materials such as explosives, toxins, or pathogens. Other biological sensors can be used to improve world health by providing timely diagnostics and applying corrective measures to the human body. Electro-microfluidic packaging can easily represent over fifty percent of the product cost and, as with Integrated Circuits (IC), the industry should evolve to standard packaging solutions. Standard packaging schemes will minimize cost and bring products to market sooner.

  11. Microfluidic perfusion culture.

    PubMed

    Hattori, Koji; Sugiura, Shinji; Kanamori, Toshiyuki

    2014-01-01

    Microfluidic perfusion culture is a novel technique to culture animal cells in a small-scale microchamber with medium perfusion. Polydimethylsiloxane (PDMS) is the most popular material to fabricate a microfluidic perfusion culture chip. Photolithography and replica molding techniques are generally used for fabrication of a microfluidic perfusion culture chip. Pressure-driven perfusion culture system is convenient technique to carry out the perfusion culture of animal cells in a microfluidic device. Here, we describe a general theory on microfluid network design, microfabrication technique, and experimental technique for pressure-driven perfusion culture in an 8 × 8 microchamber array on a glass slide-sized microchip made out of PDMS. PMID:24297421

  12. Active probes and microfluidic ink delivery for Dip Pen Nanolithography

    NASA Astrophysics Data System (ADS)

    Rosner, Bjoern; Duenas, Terrisa; Banerjee, Debjyoti; Shile, Roger; Amro, Nabil; Rendlen, Jeff

    2004-03-01

    Dip Pen Nanolithography (DPNTM) is a scanning probe technique for nanoscale lithography: A sharp tip is coated with a functional molecule (the "ink") and then brought into contact with a surface where it deposits ink via a water meniscus. The DPN process is a direct-write pattern transfer technique with nanometer resolution and is inherently general with respect to usable inks and substrates including biomolecules such as proteins and oligonucleotides. We present functional extensions of the basic DPN process by showing actuated multi-probes as well as microfluidic ink delivery. We present the fabrication process and characterization of such active probes that use the bimorph effect to induce deflection of individual cantilevers as well as the integration of these probes. We also developed the capability to write with multiple inks on the probe array permitting the fabrication of multi-component nanodevices in one writing session. For this purpose, we fabricate passive microfluidic devices and present microfluidic behavior and ink loading performance of these components.

  13. Ultrafast microfluidics using surface acoustic waves

    PubMed Central

    Yeo, Leslie Y.; Friend, James R.

    2009-01-01

    We demonstrate that surface acoustic waves (SAWs), nanometer amplitude Rayleigh waves driven at megahertz order frequencies propagating on the surface of a piezoelectric substrate, offer a powerful method for driving a host of extremely fast microfluidic actuation and micro∕bioparticle manipulation schemes. We show that sessile drops can be translated rapidly on planar substrates or fluid can be pumped through microchannels at 1–10 cm∕s velocities, which are typically one to two orders quicker than that afforded by current microfluidic technologies. Through symmetry-breaking, azimuthal recirculation can be induced within the drop to drive strong inertial microcentrifugation for micromixing and particle concentration or separation. Similar micromixing strategies can be induced in the same microchannel in which fluid is pumped with the SAW by merely changing the SAW frequency to rapidly switch the uniform through-flow into a chaotic oscillatory flow by exploiting superpositioning of the irradiated sound waves from the sidewalls of the microchannel. If the flow is sufficiently quiescent, the nodes of the transverse standing wave that arises across the microchannel also allow for particle aggregation, and hence, sorting on nodal lines. In addition, the SAW also facilitates other microfluidic capabilities. For example, capillary waves excited at the free surface of a sessile drop by the SAW underneath it can be exploited for micro∕nanoparticle collection and sorting at nodal points or lines at low powers. At higher powers, the large accelerations off the substrate surface as the SAW propagates across drives rapid destabilization of the drop free surface giving rise to inertial liquid jets that persist over 1–2 cm in length or atomization of the entire drop to produce 1–10 μm monodispersed aerosol droplets, which can be exploited for ink-jet printing, mass spectrometry interfacing, or pulmonary drug delivery. The atomization of polymer∕protein solutions

  14. Microfluidic White Organic Light-Emitting Diode Based on Integrated Patterns of Greenish-Blue and Yellow Solvent-Free Liquid Emitters

    PubMed Central

    Kobayashi, Naofumi; Kasahara, Takashi; Edura, Tomohiko; Oshima, Juro; Ishimatsu, Ryoichi; Tsuwaki, Miho; Imato, Toshihiko; Shoji, Shuichi; Mizuno, Jun

    2015-01-01

    We demonstrated a novel microfluidic white organic light-emitting diode (microfluidic WOLED) based on integrated sub-100-μm-wide microchannels. Single-μm-thick SU-8-based microchannels, which were sandwiched between indium tin oxide (ITO) anode and cathode pairs, were fabricated by photolithography and heterogeneous bonding technologies. 1-Pyrenebutyric acid 2-ethylhexyl ester (PLQ) was used as a solvent-free greenish-blue liquid emitter, while 2,8-di-tert-butyl-5,11-bis(4-tert-butylphenyl)-6,12-diphenyltetracene (TBRb)-doped PLQ was applied as a yellow liquid emitter. In order to form the liquid white light-emitting layer, the greenish-blue and yellow liquid emitters were alternately injected into the integrated microchannels. The fabricated electro-microfluidic device successfully exhibited white electroluminescence (EL) emission via simultaneous greenish-blue and yellow emissions under an applied voltage of 100 V. A white emission with Commission Internationale de l’Declairage (CIE) color coordinates of (0.40, 0.42) was also obtained; the emission corresponds to warm-white light. The proposed device has potential applications in subpixels of liquid-based microdisplays and for lighting. PMID:26439164

  15. Microfluidic White Organic Light-Emitting Diode Based on Integrated Patterns of Greenish-Blue and Yellow Solvent-Free Liquid Emitters

    NASA Astrophysics Data System (ADS)

    Kobayashi, Naofumi; Kasahara, Takashi; Edura, Tomohiko; Oshima, Juro; Ishimatsu, Ryoichi; Tsuwaki, Miho; Imato, Toshihiko; Shoji, Shuichi; Mizuno, Jun

    2015-10-01

    We demonstrated a novel microfluidic white organic light-emitting diode (microfluidic WOLED) based on integrated sub-100-μm-wide microchannels. Single-μm-thick SU-8-based microchannels, which were sandwiched between indium tin oxide (ITO) anode and cathode pairs, were fabricated by photolithography and heterogeneous bonding technologies. 1-Pyrenebutyric acid 2-ethylhexyl ester (PLQ) was used as a solvent-free greenish-blue liquid emitter, while 2,8-di-tert-butyl-5,11-bis(4-tert-butylphenyl)-6,12-diphenyltetracene (TBRb)-doped PLQ was applied as a yellow liquid emitter. In order to form the liquid white light-emitting layer, the greenish-blue and yellow liquid emitters were alternately injected into the integrated microchannels. The fabricated electro-microfluidic device successfully exhibited white electroluminescence (EL) emission via simultaneous greenish-blue and yellow emissions under an applied voltage of 100 V. A white emission with Commission Internationale de l’Declairage (CIE) color coordinates of (0.40, 0.42) was also obtained; the emission corresponds to warm-white light. The proposed device has potential applications in subpixels of liquid-based microdisplays and for lighting.

  16. Model for evaluating patterned charge regulation contribution to electrostatic interactions between proteins

    NASA Astrophysics Data System (ADS)

    Hollenbeck, Dawn; Martini, K. Michael; Langner, Andreas; Ross, David; Harkin, Anthony; Nelson, Edward; Thurston, George

    2010-03-01

    We study the pattern-specific work of charging for two spherical model proteins in close proximity in ionic solution, using a grand-canonical partition function together with a coarse-grained, linear Debye-Huckel model to calculate the needed work of charging for each possible proton occupancy configuration. We seek to delineate a parameter-space phase diagram to characterize the circumstances under which patterned charge regulation, attractions due to heterogeneous protein charging patterns, and screened net protein charge could individually dominate the electrostatic portion of the interaction between model particles. Within the model, we place titratable residues in accordance with the tertiary protein structure, as is done in the case of a single protein within the Tanford-Kirkwood protein electrostatics model. We use Monte-Carlo simulation and analytical work to evaluate how the local statistics of the charging patterns on each protein respond to close proximity and relative orientation of neighboring proteins.

  17. Two-dimensional isoelectric focusing OFFGEL and microfluidic lab-on-chip electrophoresis for assessing dissolved proteins in seawater.

    PubMed

    García-Otero, Natalia; Peña-Vázquez, Elena; Barciela-Alonso, María Carmen; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

    2013-06-18

    Dissolved proteins were assessed in surface and deep seawater by two-dimensional isoelectric focusing (IEF) OFFGEL-lab-on-chip (LOC) electrophoresis after tangential flow ultrafiltration followed by centrifugal ultrafiltration (preconcentration factor of 3000). Dissolved protein isolation was performed by treating the ultrafiltrated retentate with cold acetone and also with chloroform as precipitating reagents. The best electrophoretic behavior of the isolated proteins was obtained after protein precipitation with chloroform before different rinsing stages for removing methanol and water interferences. Metals bound to proteins in the different OFFGEL fractions were assessed by inductively coupled plasma-optical emission spectrometry and electrothermal atomic absorption spectrometry, under optimized operating conditions. Experiments regarding stability of the metal-binding proteins [superoxide dismutase (SOD) and alcohol dehydrogenase (ADH) as protein models] showed the integrity of the Zn-binding SOD/ADH under the OFFGEL electrophoretic conditions. However, stability of Cu bound to SOD is not guaranteed. The first electrophoretic dimension (IEF OFFGEL) showed that dissolved proteins in surface seawater exhibit alkaline isoelectric points (pIs of 8.10 and 8.37) and also acid Ips (4.82, 5.13, 5.43, and 5.73), while LOC showed that the isolated proteins exhibit a spread molecular weight range (within 15 - 63 kDa); although, high molecular weights were the most commonly found. Regarding deep seawater, isolated proteins were of acid Ips (from 3.30 to 4.22) and low molecular weight (within the 21-24 kDa range). Elements such as Cd, Cu, Mn, and Ni were mainly associated with dissolved proteins of alkaline pIs in surface seawater, while Zn was mainly associated to proteins of acid pIs. However, only Cu and Mn were found to be bound to dissolved proteins of higher Ips in deep seawater, and the amount of Mn (from 68 to 84 μg L(-1)) was higher than that found in dissolved

  18. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

    PubMed

    Hsiao, Tony W; Tresco, Patrick A; Hlady, Vladimir

    2015-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels.

  19. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

    PubMed

    Hsiao, Tony W; Tresco, Patrick A; Hlady, Vladimir

    2015-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels. PMID:25477179

  20. A microfluidic platform with a flow-balanced fluidic network for osteoarthritis diagnosis

    NASA Astrophysics Data System (ADS)

    Kim, Kangil; Park, Yoo Min; Yoon, Hyun C.; Yang, Sang Sik

    2013-05-01

    Osteoarthritis (OA) is one of the most common human diseases, and the occurrence of OA is likely to increase with the increase of population ages. The diagnosis of OA is based on patientrelevant measures, structural measures, and measurement of biomarkers that are released through joint metabolism. Traditionally, radiography or magnetic resonance imaging (MRI) is used to diagnose OA and predict its course. However, diagnostic imaging in OA provides only indirect information on pathology and treatment response. A sensing of OA based on the detection of biomarkers insignificantly improves the accuracy and sensitivity of diagnosis and reduces the cost compared with that of radiography or MRI. In our former study, we proposed microfluidic platform to detect biomarker of OA. But the platform can detect only one biomarker because it has one microfluidic channel. In this report, we proposes microfluidic platform that can detect several biomarkers. The proposed platform has three layers. The bottom layer has gold patterns on a Si substrate for optical sensing. The middle layer and top layer were fabricated by polydimethysiloxane (PDMS) using soft-lithography. The middle layer has four channels connecting top layer to bottom layer. The top layer consists of one sample injection inlet, and four antibody injection inlets. To this end, we designed a flow-balanced microfluidic network using analogy between electric and hydraulic systems. Also, the designed microfluidic network was confirmed by finite element model (FEM) analysis using COMSOL FEMLAB. To verify the efficiency of fabricated platform, the optical sensing test was performed to detect biomarker of OA using fluorescence microscope. We used cartilage oligomeric matrix protein (COMP) as biomarker because it reflects specific changes in joint tissues. The platform successfully detected various concentration of COMP (0, 100, 500, 1000 ng/ml) at each chamber. The effectiveness of the microfluidic platform was verified

  1. Digital Microfluidics Sample Analyzer

    NASA Technical Reports Server (NTRS)

    Pollack, Michael G.; Srinivasan, Vijay; Eckhardt, Allen; Paik, Philip Y.; Sudarsan, Arjun; Shenderov, Alex; Hua, Zhishan; Pamula, Vamsee K.

    2010-01-01

    Three innovations address the needs of the medical world with regard to microfluidic manipulation and testing of physiological samples in ways that can benefit point-of-care needs for patients such as premature infants, for which drawing of blood for continuous tests can be life-threatening in their own right, and for expedited results. A chip with sample injection elements, reservoirs (and waste), droplet formation structures, fluidic pathways, mixing areas, and optical detection sites, was fabricated to test the various components of the microfluidic platform, both individually and in integrated fashion. The droplet control system permits a user to control droplet microactuator system functions, such as droplet operations and detector operations. Also, the programming system allows a user to develop software routines for controlling droplet microactuator system functions, such as droplet operations and detector operations. A chip is incorporated into the system with a controller, a detector, input and output devices, and software. A novel filler fluid formulation is used for the transport of droplets with high protein concentrations. Novel assemblies for detection of photons from an on-chip droplet are present, as well as novel systems for conducting various assays, such as immunoassays and PCR (polymerase chain reaction). The lab-on-a-chip (a.k.a., lab-on-a-printed-circuit board) processes physiological samples and comprises a system for automated, multi-analyte measurements using sub-microliter samples of human serum. The invention also relates to a diagnostic chip and system including the chip that performs many of the routine operations of a central labbased chemistry analyzer, integrating, for example, colorimetric assays (e.g., for proteins), chemiluminescence/fluorescence assays (e.g., for enzymes, electrolytes, and gases), and/or conductometric assays (e.g., for hematocrit on plasma and whole blood) on a single chip platform.

  2. Trehalose Glycopolymer Resists Allow Direct Writing of Protein Patterns by Electron-Beam Lithography

    PubMed Central

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y.; Maynard, Heather D.

    2015-01-01

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein we present a new resist that protects proteins during electron beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively cross-link to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometer and nanometer scale without requiring cleanroom conditions. PMID:25791943

  3. Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography

    NASA Astrophysics Data System (ADS)

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y.; Maynard, Heather D.

    2015-03-01

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein, we present a new resist that protects proteins during electron-beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively crosslink to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron-beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high-precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometre and nanometre scale without requiring cleanroom conditions.

  4. Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography.

    PubMed

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y; Maynard, Heather D

    2015-03-20

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein, we present a new resist that protects proteins during electron-beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively crosslink to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron-beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high-precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometre and nanometre scale without requiring cleanroom conditions.

  5. Patterned polymer nanowire arrays as an effective protein immobilizer for biosensing and HIV detection

    NASA Astrophysics Data System (ADS)

    Shen, Yue; Liu, Yingyi; Zhu, Guang; Fang, Hao; Huang, Yunhui; Jiang, Xingyu; Wang, Zhong L.

    2012-12-01

    We report an array of polymeric nanowires for effectively immobilizing biomolecules on biochips owing to the large surface area. The nanowires were fabricated in predesigned patterns using an inductively coupled plasma (ICP) etching process. Microfluidic biochips integrated using the substrates with arrays of nanowires and polydimethylsiloxane channels have been demonstrated to be effective for detecting antigens, and a detection limit of antigens at 0.2 μg mL-1 has been achieved, which is improved by a factor of 50 compared to that based on flat substrates without the nanowires. In addition, the high sensitivity for clinical detection of human immunodeficiency virus (HIV) antibody has also been demonstrated, showing a 20 times enhancement in fluorescent signal intensity between the samples with positive and negative HIV.

  6. Easy Fabrication of Thin Membranes with Through Holes. Application to Protein Patterning

    PubMed Central

    Arasi, Bakya; Gauthier, Nils; Viasnoff, Virgile

    2012-01-01

    Since protein patterning on 2D surfaces has emerged as an important tool in cell biology, the development of easy patterning methods has gained importance in biology labs. In this paper we present a simple, rapid and reliable technique to fabricate thin layers of UV curable polymer with through holes. These membranes are as easy to fabricate as microcontact printing stamps and can be readily used for stencil patterning. We show how this microfabrication scheme allows highly reproducible and highly homogeneous protein patterning with micron sized resolution on surfaces as large as 10 cm2. Using these stencils, fragile proteins were patterned without loss of function in a fully hydrated state. We further demonstrate how intricate patterns of multiple proteins can be achieved by stacking the stencil membranes. We termed this approach microserigraphy. PMID:22952944

  7. Easy fabrication of thin membranes with through holes. Application to protein patterning.

    PubMed

    Masters, Thomas; Engl, Wilfried; Weng, Zhe L; Arasi, Bakya; Gauthier, Nils; Viasnoff, Virgile

    2012-01-01

    Since protein patterning on 2D surfaces has emerged as an important tool in cell biology, the development of easy patterning methods has gained importance in biology labs. In this paper we present a simple, rapid and reliable technique to fabricate thin layers of UV curable polymer with through holes. These membranes are as easy to fabricate as microcontact printing stamps and can be readily used for stencil patterning. We show how this microfabrication scheme allows highly reproducible and highly homogeneous protein patterning with micron sized resolution on surfaces as large as 10 cm(2). Using these stencils, fragile proteins were patterned without loss of function in a fully hydrated state. We further demonstrate how intricate patterns of multiple proteins can be achieved by stacking the stencil membranes. We termed this approach microserigraphy. PMID:22952944

  8. Micro-fluidic interconnect

    DOEpatents

    Okandan, Murat; Galambos, Paul C.; Benavides, Gilbert L.; Hetherington, Dale L.

    2006-02-28

    An apparatus for simultaneously aligning and interconnecting microfluidic ports is presented. Such interconnections are required to utilize microfluidic devices fabricated in Micro-Electromechanical-Systems (MEMS) technologies, that have multiple fluidic access ports (e.g. 100 micron diameter) within a small footprint, (e.g. 3 mm.times.6 mm). Fanout of the small ports of a microfluidic device to a larger diameter (e.g. 500 microns) facilitates packaging and interconnection of the microfluidic device to printed wiring boards, electronics packages, fluidic manifolds etc.

  9. Microfluidics for manipulating cells.

    PubMed

    Mu, Xuan; Zheng, Wenfu; Sun, Jiashu; Zhang, Wei; Jiang, Xingyu

    2013-01-14

    Microfluidics, a toolbox comprising methods for precise manipulation of fluids at small length scales (micrometers to millimeters), has become useful for manipulating cells. Its uses range from dynamic management of cellular interactions to high-throughput screening of cells, and to precise analysis of chemical contents in single cells. Microfluidics demonstrates a completely new perspective and an excellent practical way to manipulate cells for solving various needs in biology and medicine. This review introduces and comments on recent achievements and challenges of using microfluidics to manipulate and analyze cells. It is believed that microfluidics will assume an even greater role in the mechanistic understanding of cell biology and, eventually, in clinical applications.

  10. Parallel Imaging Microfluidic Cytometer

    PubMed Central

    Ehrlich, Daniel J.; McKenna, Brian K.; Evans, James G.; Belkina, Anna C.; Denis, Gerald V.; Sherr, David; Cheung, Man Ching

    2011-01-01

    By adding an additional degree of freedom from multichannel flow, the parallel microfluidic cytometer (PMC) combines some of the best features of flow cytometry (FACS) and microscope-based high-content screening (HCS). The PMC (i) lends itself to fast processing of large numbers of samples, (ii) adds a 1-D imaging capability for intracellular localization assays (HCS), (iii) has a high rare-cell sensitivity and, (iv) has an unusual capability for time-synchronized sampling. An inability to practically handle large sample numbers has restricted applications of conventional flow cytometers and microscopes in combinatorial cell assays, network biology, and drug discovery. The PMC promises to relieve a bottleneck in these previously constrained applications. The PMC may also be a powerful tool for finding rare primary cells in the clinic. The multichannel architecture of current PMC prototypes allows 384 unique samples for a cell-based screen to be read out in approximately 6–10 minutes, about 30-times the speed of most current FACS systems. In 1-D intracellular imaging, the PMC can obtain protein localization using HCS marker strategies at many times the sample throughput of CCD-based microscopes or CCD-based single-channel flow cytometers. The PMC also permits the signal integration time to be varied over a larger range than is practical in conventional flow cytometers. The signal-to-noise advantages are useful, for example, in counting rare positive cells in the most difficult early stages of genome-wide screening. We review the status of parallel microfluidic cytometry and discuss some of the directions the new technology may take. PMID:21704835

  11. Microfluidic devices for cell cultivation and proliferation

    PubMed Central

    Tehranirokh, Masoomeh; Kouzani, Abbas Z.; Francis, Paul S.; Kanwar, Jagat R.

    2013-01-01

    Microfluidic technology provides precise, controlled-environment, cost-effective, compact, integrated, and high-throughput microsystems that are promising substitutes for conventional biological laboratory methods. In recent years, microfluidic cell culture devices have been used for applications such as tissue engineering, diagnostics, drug screening, immunology, cancer studies, stem cell proliferation and differentiation, and neurite guidance. Microfluidic technology allows dynamic cell culture in microperfusion systems to deliver continuous nutrient supplies for long term cell culture. It offers many opportunities to mimic the cell-cell and cell-extracellular matrix interactions of tissues by creating gradient concentrations of biochemical signals such as growth factors, chemokines, and hormones. Other applications of cell cultivation in microfluidic systems include high resolution cell patterning on a modified substrate with adhesive patterns and the reconstruction of complicated tissue architectures. In this review, recent advances in microfluidic platforms for cell culturing and proliferation, for both simple monolayer (2D) cell seeding processes and 3D configurations as accurate models of in vivo conditions, are examined. PMID:24273628

  12. Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks.

    PubMed

    Chiva, M; Saperas, N; Ribes, E

    2011-12-01

    In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and

  13. Unconventional microfluidics: expanding the discipline

    PubMed Central

    Nawaz, Ahmad Ahsan; Mao, Xiaole; Stratton, Zackary S.; Huang, Tony Jun

    2014-01-01

    Since its inception, the discipline of microfluidics has been harnessed for innovations in the biomedicine/chemistry fields—and to great effect. This success has had the natural side-effect of stereotyping microfluidics as a platform for medical diagnostics and miniaturized lab processes. But microfluidics has more to offer. And very recently, some researchers have successfully applied microfluidics to fields outside its traditional domains. In this Focus article, we highlight notable examples of such “unconventional” microfluidics applications (e.g., robotics, electronics). It is our hope that these early successes in unconventional microfluidics prompt further creativity, and inspire readers to expand the microfluidics discipline. PMID:23478651

  14. Unconventional microfluidics: expanding the discipline.

    PubMed

    Nawaz, Ahmad Ahsan; Mao, Xiaole; Stratton, Zackary S; Huang, Tony Jun

    2013-04-21

    Since its inception, the discipline of microfluidics has been harnessed for innovations in the biomedicine/chemistry fields-and to great effect. This success has had the natural side-effect of stereotyping microfluidics as a platform for medical diagnostics and miniaturized lab processes. But microfluidics has more to offer. And very recently, some researchers have successfully applied microfluidics to fields outside its traditional domains. In this Focus article, we highlight notable examples of such "unconventional" microfluidics applications (e.g., robotics, electronics). It is our hope that these early successes in unconventional microfluidics prompt further creativity, and inspire readers to expand the microfluidics discipline.

  15. Effect of hair dyes and bleach on the hair protein patterns as revealed by isoelectric focusing.

    PubMed

    Nagai, A; Komoriya, H; Bunai, Y; Yamada, S; Jiang, X Y; Ohya, I

    1991-06-01

    The effect of hair dyes, i.e., temporary, semi-permanent, or permanent hair dyes, or hair bleach on the isoelectric focusing (IEF) hair protein patterns was studied. A permanent hair dye (metallic, alkaline oxidative, or acidic oxidative) and hair bleach induced changes in the IEF hair protein patterns and in the intensity of hair protein bands. The changes in the IEF patterns, caused by the alkaline oxidative dye or the bleach, are considered to result from the combined effect of an alkaline agent and an oxidative agent in the alkaline oxidative dye and in the hair bleach.

  16. Effect of hair dyes and bleach on the hair protein patterns as revealed by isoelectric focusing.

    PubMed

    Nagai, A; Komoriya, H; Bunai, Y; Yamada, S; Jiang, X Y; Ohya, I

    1991-06-01

    The effect of hair dyes, i.e., temporary, semi-permanent, or permanent hair dyes, or hair bleach on the isoelectric focusing (IEF) hair protein patterns was studied. A permanent hair dye (metallic, alkaline oxidative, or acidic oxidative) and hair bleach induced changes in the IEF hair protein patterns and in the intensity of hair protein bands. The changes in the IEF patterns, caused by the alkaline oxidative dye or the bleach, are considered to result from the combined effect of an alkaline agent and an oxidative agent in the alkaline oxidative dye and in the hair bleach. PMID:1889397

  17. PatternQuery: web application for fast detection of biomacromolecular structural patterns in the entire Protein Data Bank.

    PubMed

    Sehnal, David; Pravda, Lukáš; Svobodová Vařeková, Radka; Ionescu, Crina-Maria; Koča, Jaroslav

    2015-07-01

    Well defined biomacromolecular patterns such as binding sites, catalytic sites, specific protein or nucleic acid sequences, etc. precisely modulate many important biological phenomena. We introduce PatternQuery, a web-based application designed for detection and fast extraction of such patterns. The application uses a unique query language with Python-like syntax to define the patterns that will be extracted from datasets provided by the user, or from the entire Protein Data Bank (PDB). Moreover, the database-wide search can be restricted using a variety of criteria, such as PDB ID, resolution, and organism of origin, to provide only relevant data. The extraction generally takes a few seconds for several hundreds of entries, up to approximately one hour for the whole PDB. The detected patterns are made available for download to enable further processing, as well as presented in a clear tabular and graphical form directly in the browser. The unique design of the language and the provided service could pave the way towards novel PDB-wide analyses, which were either difficult or unfeasible in the past. The application is available free of charge at http://ncbr.muni.cz/PatternQuery. PMID:26013810

  18. PatternQuery: web application for fast detection of biomacromolecular structural patterns in the entire Protein Data Bank.

    PubMed

    Sehnal, David; Pravda, Lukáš; Svobodová Vařeková, Radka; Ionescu, Crina-Maria; Koča, Jaroslav

    2015-07-01

    Well defined biomacromolecular patterns such as binding sites, catalytic sites, specific protein or nucleic acid sequences, etc. precisely modulate many important biological phenomena. We introduce PatternQuery, a web-based application designed for detection and fast extraction of such patterns. The application uses a unique query language with Python-like syntax to define the patterns that will be extracted from datasets provided by the user, or from the entire Protein Data Bank (PDB). Moreover, the database-wide search can be restricted using a variety of criteria, such as PDB ID, resolution, and organism of origin, to provide only relevant data. The extraction generally takes a few seconds for several hundreds of entries, up to approximately one hour for the whole PDB. The detected patterns are made available for download to enable further processing, as well as presented in a clear tabular and graphical form directly in the browser. The unique design of the language and the provided service could pave the way towards novel PDB-wide analyses, which were either difficult or unfeasible in the past. The application is available free of charge at http://ncbr.muni.cz/PatternQuery.

  19. Differentiation of Campylobacter species by protein banding patterns in polyacrylamide slab gels.

    PubMed

    Ferguson, D A; Lambe, D W

    1984-09-01

    Soluble protein extracts of 37 catalase-positive strains of Campylobacter species were examined by polyacrylamide slab gel electrophoresis (PAGE). Electrophoretic banding patterns showed good correlation with biochemical tests and with available DNA homology data in distinguishing species of Campylobacter but did not differentiate subspecies or biotypes. PAGE patterns indicated that Campylobacter coli is a distinct species. Furthermore, the PAGE patterns indicated that C. jejuni and nalidixic acid-resistant thermophilic Campylobacter species (C. laridis) are each distinct species. The protein banding patterns of C. fetus subsp. venerealis and C. fetus subsp. fetus strains were distinctly different from those of the three thermophilic species. PMID:6490829

  20. Two-dimensional Protein Patterns in Labronema, Aporcelaimellus, and Eudorylaimus (Nematoda: Dorylaimida)

    PubMed Central

    Ferris, V. R.; Ferris, J. M.; Murdock, L. L.; Faghihi, J.

    1987-01-01

    Two-dimensional polyacrylamide gel electrophoretic patterns of proteins for two isolates of Labronema from Indiana were nearly identical to the pattern for L. vulvapapillatum from Europe. The pattern for a nominal isolate of L. pacificum from Florida was very different from the patterns of nominal L. pacificum isolates from Hawaii and Fiji (which had patterns very similar to each other). Patterns for four other isolates (in Eudorylaimus and Aporcelaimellus) were different from the Labronema patterns and from each other, although some constellations of protein spots were shared among all the isolates. The study demonstrates the utility of 2-D PAGE for clarifying taxonomic problems that cannot be resolved using classical morphological data alone. PMID:19290167

  1. Accelerated Biofluid Filling in Complex Microfluidic Networks by Vacuum-Pressure Accelerated Movement (V-PAM).

    PubMed

    Yu, Zeta Tak For; Cheung, Mei Ki; Liu, Shirley Xiaosu; Fu, Jianping

    2016-09-01

    Rapid fluid transport and exchange are critical operations involved in many microfluidic applications. However, conventional mechanisms used for driving fluid transport in microfluidics, such as micropumping and high pressure, can be inaccurate and difficult for implementation for integrated microfluidics containing control components and closed compartments. Here, a technology has been developed termed Vacuum-Pressure Accelerated Movement (V-PAM) capable of significantly enhancing biofluid transport in complex microfluidic environments containing dead-end channels and closed chambers. Operation of the V-PAM entails a pressurized fluid loading into microfluidic channels where gas confined inside can rapidly be dissipated through permeation through a thin, gas-permeable membrane sandwiched between microfluidic channels and a network of vacuum channels. Effects of different structural and operational parameters of the V-PAM for promoting fluid filling in microfluidic environments have been studied systematically. This work further demonstrates the applicability of V-PAM for rapid filling of temperature-sensitive hydrogels and unprocessed whole blood into complex irregular microfluidic networks such as microfluidic leaf venation patterns and blood circulatory systems. Together, the V-PAM technology provides a promising generic microfluidic tool for advanced fluid control and transport in integrated microfluidics for different microfluidic diagnosis, organs-on-chips, and biomimetic studies. PMID:27409528

  2. Application of Gap-Constraints Given Sequential Frequent Pattern Mining for Protein Function Prediction

    PubMed Central

    Park, Hyeon Ah; Kim, Taewook; Li, Meijing; Shon, Ho Sun; Park, Jeong Seok; Ryu, Keun Ho

    2015-01-01

    Objectives Predicting protein function from the protein–protein interaction network is challenging due to its complexity and huge scale of protein interaction process along with inconsistent pattern. Previously proposed methods such as neighbor counting, network analysis, and graph pattern mining has predicted functions by calculating the rules and probability of patterns inside network. Although these methods have shown good prediction, difficulty still exists in searching several functions that are exceptional from simple rules and patterns as a result of not considering the inconsistent aspect of the interaction network. Methods In this article, we propose a novel approach using the sequential pattern mining method with gap-constraints. To overcome the inconsistency problem, we suggest frequent functional patterns to include every possible functional sequence—including patterns for which search is limited by the structure of connection or level of neighborhood layer. We also constructed a tree-graph with the most crucial interaction information of the target protein, and generated candidate sets to assign by sequential pattern mining allowing gaps. Results The parameters of pattern length, maximum gaps, and minimum support were given to find the best setting for the most accurate prediction. The highest accuracy rate was 0.972, which showed better results than the simple neighbor counting approach and link-based approach. Conclusion The results comparison with other approaches has confirmed that the proposed approach could reach more function candidates that previous methods could not obtain. PMID:25938021

  3. Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

    SciTech Connect

    Valley, Cary T.; Porter, Douglas F.; Qiu, Chen; Campbell, Zachary T.; Tanaka Hall, Traci M.; Wickens, Marvin

    2012-06-28

    mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites. Despite this conserved 'two-handed' pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.

  4. Invariant patterns in crystal lattices: Implications for protein folding algorithms

    SciTech Connect

    HART,WILLIAM E.; ISTRAIL,SORIN

    2000-06-01

    Crystal lattices are infinite periodic graphs that occur naturally in a variety of geometries and which are of fundamental importance in polymer science. Discrete models of protein folding use crystal lattices to define the space of protein conformations. Because various crystal lattices provide discretizations of the same physical phenomenon, it is reasonable to expect that there will exist invariants across lattices related to fundamental properties of the protein folding process. This paper considers whether performance-guaranteed approximability is such an invariant for HP lattice models. The authors define a master approximation algorithm that has provable performance guarantees provided that a specific sublattice exists within a given lattice. They describe a broad class of crystal lattices that are approximable, which further suggests that approximability is a general property of HP lattice models.

  5. Protein patterns of black fungi under simulated Mars-like conditions.

    PubMed

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-05-29

    Two species of microcolonial fungi - Cryomyces antarcticus and Knufia perforans - and a species of black yeasts-Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.

  6. Protein patterns of black fungi under simulated Mars-like conditions

    NASA Astrophysics Data System (ADS)

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-05-01

    Two species of microcolonial fungi - Cryomyces antarcticus and Knufia perforans - and a species of black yeasts-Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.

  7. Protein patterns of black fungi under simulated Mars-like conditions

    PubMed Central

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-01-01

    Two species of microcolonial fungi – Cryomyces antarcticus and Knufia perforans - and a species of black yeasts–Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure. PMID:24870977

  8. Protein patterns of black fungi under simulated Mars-like conditions.

    PubMed

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-01-01

    Two species of microcolonial fungi - Cryomyces antarcticus and Knufia perforans - and a species of black yeasts-Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure. PMID:24870977

  9. Stem cells in microfluidics

    PubMed Central

    Wu, Huei-Wen; Lin, Chun-Che; Lee, Gwo-Bin

    2011-01-01

    Microfluidic techniques have been recently developed for cell-based assays. In microfluidic systems, the objective is for these microenvironments to mimic in vivo surroundings. With advantageous characteristics such as optical transparency and the capability for automating protocols, different types of cells can be cultured, screened, and monitored in real time to systematically investigate their morphology and functions under well-controlled microenvironments in response to various stimuli. Recently, the study of stem cells using microfluidic platforms has attracted considerable interest. Even though stem cells have been studied extensively using bench-top systems, an understanding of their behavior in in vivo-like microenvironments which stimulate cell proliferation and differentiation is still lacking. In this paper, recent cell studies using microfluidic systems are first introduced. The various miniature systems for cell culture, sorting and isolation, and stimulation are then systematically reviewed. The main focus of this review is on papers published in recent years studying stem cells by using microfluidic technology. This review aims to provide experts in microfluidics an overview of various microfluidic systems for stem cell research. PMID:21522491

  10. Microfluidics and microbial engineering.

    PubMed

    Kou, Songzi; Cheng, Danhui; Sun, Fei; Hsing, I-Ming

    2016-02-01

    The combination of microbial engineering and microfluidics is synergistic in nature. For example, microfluidics is benefiting from the outcome of microbial engineering and many reported point-of-care microfluidic devices employ engineered microbes as functional parts for the microsystems. In addition, microbial engineering is facilitated by various microfluidic techniques, due to their inherent strength in high-throughput screening and miniaturization. In this review article, we firstly examine the applications of engineered microbes for toxicity detection, biosensing, and motion generation in microfluidic platforms. Secondly, we look into how microfluidic technologies facilitate the upstream and downstream processes of microbial engineering, including DNA recombination, transformation, target microbe selection, mutant characterization, and microbial function analysis. Thirdly, we highlight an emerging concept in microbial engineering, namely, microbial consortium engineering, where the behavior of a multicultural microbial community rather than that of a single cell/species is delineated. Integrating the disciplines of microfluidics and microbial engineering opens up many new opportunities, for example in diagnostics, engineering of microbial motors, development of portable devices for genetics, high throughput characterization of genetic mutants, isolation and identification of rare/unculturable microbial species, single-cell analysis with high spatio-temporal resolution, and exploration of natural microbial communities.

  11. Different evolutionary patterns of classical swine fever virus envelope proteins.

    PubMed

    Li, Yan; Yang, Zexiao; Zhang, Mingwang

    2016-03-01

    Classical swine fever virus (CSFV) is the causative agent of classical swine fever, which is a highly contagious disease of the domestic pig as well as wild boar. The proteins E(rns), E1, and E2 are components of the viral envelope membrane. They are also implicated in virus attachment and entry, replication, and (or) anti-immune response. Here, we studied the genetic variations of these envelope proteins in the evolution of CSFV. The results reveal that the envelope proteins underwent different evolutionary fates. In E(rns) and E1, but not E2, a number of amino acid sites experienced functional divergence. Furthermore, the diversification in E(rns) and E1 was generally episodic because the divergence-related changes of E1 only occurred with the separation of 2 major groups of CSFV and that of E(rns) took place with the division of 1 major group. The major divergence-related sites of E(rns) are located on one of the substrate-binding regions of the RNase domain and C-terminal extension. These functional domains have been reported to block activation of the innate immune system and attachment and entry into host cells, respectively. Our results may shed some light on the divergent roles of the envelope proteins. PMID:26911308

  12. In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana

    PubMed Central

    Urbanus, Susan L; de Folter, Stefan; Shchennikova, Anna V; Kaufmann, Kerstin; Immink, Richard GH; Angenent, Gerco C

    2009-01-01

    Background MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). Results We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. Conclusion This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during

  13. A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels

    NASA Astrophysics Data System (ADS)

    Deforest, Cole A.; Tirrell, David A.

    2015-05-01

    Although biochemically patterned hydrogels are capable of recapitulating many critical aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomolecular signalling cues in such systems has proved difficult. Here, we demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection-oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, we accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. Our protein-patterning approach should provide further avenues to probe and direct changes in cell physiology in response to dynamic biochemical signalling.

  14. Quantity of dietary protein intake, but not pattern of intake, affects net protein balance primarily through differences in protein synthesis in older adults.

    PubMed

    Kim, Il-Young; Schutzler, Scott; Schrader, Amy; Spencer, Horace; Kortebein, Patrick; Deutz, Nicolaas E P; Wolfe, Robert R; Ferrando, Arny A

    2015-01-01

    To examine whole body protein turnover and muscle protein fractional synthesis rate (MPS) following ingestions of protein in mixed meals at two doses of protein and two intake patterns, 20 healthy older adult subjects (52-75 yr) participated in one of four groups in a randomized clinical trial: a level of protein intake of 0.8 g (1RDA) or 1.5 g·kg(-1)·day(-1) (∼2RDA) with uneven (U: 15/20/65%) or even distribution (E: 33/33/33%) patterns of intake for breakfast, lunch, and dinner over the day (1RDA-U, 1RDA-E, 2RDA-U, or 2RDA-E). Subjects were studied with primed continuous infusions of L-[(2)H5]phenylalanine and L-[(2)H2]tyrosine on day 4 following 3 days of diet habituation. Whole body protein kinetics [protein synthesis (PS), breakdown, and net balance (NB)] were expressed as changes from the fasted to the fed states. Positive NB was achieved at both protein levels, but NB was greater in 2RDA vs. 1RDA (94.8 ± 6.0 vs. 58.9 ± 4.9 g protein/750 min; P = 0.0001), without effects of distribution on NB. The greater NB was due to the higher PS with 2RDA vs. 1RDA (15.4 ± 4.8 vs. -18.0 ± 8.4 g protein/750 min; P = 0.0018). Consistent with PS, MPS was greater with 2RDA vs. 1RDA, regardless of distribution patterns. In conclusion, whole body net protein balance was greater with protein intake above recommended dietary allowance (0.8 g protein·kg(-1)·day(-1)) in the context of mixed meals, without demonstrated effects of protein intake pattern, primarily through higher rates of protein synthesis at whole body and muscle levels.

  15. Nanomaterials meet microfluidics.

    PubMed

    Pumera, Martin

    2011-05-28

    Nanomaterials and lab-on-a-chip platforms have undergone enormous development during the past decade. Here, we present an overview of how microfluidics benefited from the use of nanomaterials for the enhanced separation and detection of analytes. We also discuss how nanomaterials benefit from microfluidics in terms of synthesis and in terms of the simulation of environments for nanomotors and nanorobots. In our opinion, the "marriage" of nanomaterials and microfluidics is highly beneficial and is expected to solve vital challenges in related fields.

  16. Polyurethane-based microfluidic devices for blood contacting applications.

    PubMed

    Wu, Wen-I; Sask, Kyla N; Brash, John L; Selvaganapathy, P Ravi

    2012-03-01

    Protein adsorption on PDMS surfaces poses a significant challenge in microfluidic devices that come into contact with biofluids such as blood. Polyurethane (PU) is often used for the construction of medical devices, but despite having several attractive properties for biointerfacing, it has not been widely used in microfluidic devices. In this work we developed two new fabrication processes for making thin, transparent and flexible PU-based microfluidic devices. Methods for the fabrication and bonding of microchannels, the integration of fluidic interconnections and surface modification with hydrophilic polyethylene oxide (PEO) to reduce protein adsorption are detailed. Using these processes, microchannels were produced having high transparency (96% that of glass in visible light), high bond strength (326.4 kPa) and low protein adsorption (80% reduction in fibrinogen adsorption vs. unmodified PDMS), which is critical for prevention of fouling. Our findings indicate that PEO modified PU could serve as an effective alternative to PDMS in blood contacting microfluidic applications.

  17. Screening of biomineralization using microfluidics.

    PubMed

    Yin, Huabing; Ji, Bozhi; Dobson, Phillip S; Mosbahi, Khedidja; Glidle, Andrew; Gadegaard, Nikolaj; Freer, Andy; Cooper, Jonathan M; Cusack, Maggie

    2009-01-01

    Biomineralization is the process where biological systems produce well-defined composite structures such as shell, teeth, and bones. Currently, there is substantial momentum to investigate the processes implicated in biomineralization and to unravel the complex roles of proteins in the control of polymorph switching. An understanding of these processes may have wide-ranging significance in health care applications and in the development of advanced materials. We have demonstrated a microfluidic approach toward these challenges. A reversibly sealed T-junction microfluidic device was fabricated to investigate the influence of extrapallial (EP) fluid proteins in polymorph control of crystal formation in mollusk shells. A range of conditions were investigated on chip, allowing fast screening of various combinations of ion, pH, and protein concentrations. The dynamic formation of crystals was monitored on chip and combined with in situ Raman to reveal the polymorph in real time. To this end, we have demonstrated the unique advantages of this integrated approach in understanding the processes involved in biomineralization and revealing information that is impossible to obtain using traditional methods.

  18. A Novel Technique for Micro-patterning Proteins and Cells on Polyacrylamide Gels

    PubMed Central

    Tang, Xin; Ali, M. Yakut; Saif, M. Taher A.

    2012-01-01

    Spatial patterning of proteins (extracellular matrix, ECM) for living cells on polyacrylamide (PA) hydrogels has been technically challenging due to the compliant nature of the hydrogels and their aqueous environment. Traditional micro-fabrication process is not applicable. Here we report a simple, novel and general method to pattern a variety of commonly used cell adhesion molecules, i.e. Fibronectin (FN), Laminin (LN) and Collagen I (CN), etc. on PA gels. The pattern is first printed on a hydrophilic glass using polydimethylsiloxane (PDMS) stamp and micro-contact printing (μCP). Pre-polymerization solution is applied on the patterned glass and is then sandwiched by a functionalized glass slide, which covalently binds to the gel. The hydrophilic glass slide is then peeled off from the gel when the protein patterns detach from the glass, but remain intact with the gel. The pattern is thus transferred to the gel. The mechanism of pattern transfer is studied in light of interfacial mechanics. It is found that hydrophilic glass offers strong enough adhesion with ECM proteins such that a pattern can be printed, but weak enough adhesion such that they can be completely peeled off by the polymerized gel. This balance is essential for successful pattern transfer. As a demonstration, lines of FN, LN and CN with widths varying from 5–400 μm are patterned on PA gels. Normal fibroblasts (MKF) are cultured on the gel surfaces. The cell attachment and proliferation are confined within these patterns. The method avoids the use of any toxic chemistry often used to pattern different proteins on gel surfaces. PMID:23002394

  19. High-quality combinatorial protein libraries using the binary patterning approach.

    PubMed

    Bradley, Luke H

    2014-01-01

    Protein combinatorial libraries have become a platform technology for exploring protein sequence space for novel molecules for use in research, synthetic biology, biotechnology, and medicine. To expedite the isolation of proteins with novel/desired functions using screens and selections, high-quality approaches that generate protein libraries rich in folded and soluble structures are desirable for this goal. The binary patterning approach is a protein library design method that incorporates elements of both rational design and combinatorial diversity to specify the arrangement of polar and nonpolar amino acid residues in the context of a desired, folded tertiary structure template. An overview of the considerations necessary to design and construct binary patterned libraries of de novo and natural proteins is presented.

  20. Integrated reactive ion etching to pattern cross-linked hydrophilic polymer structures for protein immobilization

    NASA Astrophysics Data System (ADS)

    Bhatnagar, Parijat; Strickland, Aaron D.; Kim, Il; Malliaras, George G.; Batt, Carl A.

    2007-04-01

    Patterning of cross-linked hydrophilic polymer features using reactive ion etching (RIE) capable of covalently immobilizing proteins has been achieved. Projection photolithography was used to pattern photoresist to create micromolds. Vapor phase molecular self-assembly of polymerizable monolayer in molds allowed covalent binding of hydrogel on surface during free-radical polymerization. Excess hydrogel blanket film was consumed with oxygen RIE resulting into hydrogel pattern of 1μm size aligned to prefabricated silicon oxide structures. Proteins were finally coupled through their primary amine groups selectively to acid functionalized hydrogel features through stable amide linkages using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide.

  1. Creating two-dimensional patterned substrates for protein and cell confinement.

    PubMed

    Johnson, Dawn M; LaFranzo, Natalie A; Maurer, Joshua A

    2011-01-01

    Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.(1) While microcontact printing can be employed to directly print a large number of molecules, including proteins,(2) DNA,(3) and silanes,(4) the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.(5) This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern. The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.(6) These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior(7) to the creation of microelectronics.(8) While other types of monolayer chemistry have been used for cell culture studies, including work from

  2. Microfluidics: reframing biological enquiry.

    PubMed

    Duncombe, Todd A; Tentori, Augusto M; Herr, Amy E

    2015-09-01

    The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools - in conjunction with advanced imaging, bioinformatics and molecular biology approaches - will transform biology into a precision science.

  3. Microfluidic chemical reaction circuits

    DOEpatents

    Lee, Chung-cheng; Sui, Guodong; Elizarov, Arkadij; Kolb, Hartmuth C.; Huang, Jiang; Heath, James R.; Phelps, Michael E.; Quake, Stephen R.; Tseng, Hsian-rong; Wyatt, Paul; Daridon, Antoine

    2012-06-26

    New microfluidic devices, useful for carrying out chemical reactions, are provided. The devices are adapted for on-chip solvent exchange, chemical processes requiring multiple chemical reactions, and rapid concentration of reagents.

  4. Cell manipulation in microfluidics.

    PubMed

    Yun, Hoyoung; Kim, Kisoo; Lee, Won Gu

    2013-06-01

    Recent advances in the lab-on-a-chip field in association with nano/microfluidics have been made for new applications and functionalities to the fields of molecular biology, genetic analysis and proteomics, enabling the expansion of the cell biology field. Specifically, microfluidics has provided promising tools for enhancing cell biological research, since it has the ability to precisely control the cellular environment, to easily mimic heterogeneous cellular environment by multiplexing, and to analyze sub-cellular information by high-contents screening assays at the single-cell level. Various cell manipulation techniques in microfluidics have been developed in accordance with specific objectives and applications. In this review, we examine the latest achievements of cell manipulation techniques in microfluidics by categorizing externally applied forces for manipulation: (i) optical, (ii) magnetic, (iii) electrical, (iv) mechanical and (v) other manipulations. We furthermore focus on history where the manipulation techniques originate and also discuss future perspectives with key examples where available.

  5. Codon Bias Patterns of E. coli’s Interacting Proteins

    PubMed Central

    Dilucca, Maddalena; Cimini, Giulio; Semmoloni, Andrea; Deiana, Antonio; Giansanti, Andrea

    2015-01-01

    Synonymous codons, i.e., DNA nucleotide triplets coding for the same amino acid, are used differently across the variety of living organisms. The biological meaning of this phenomenon, known as codon usage bias, is still controversial. In order to shed light on this point, we propose a new codon bias index, CompAI, that is based on the competition between cognate and near-cognate tRNAs during translation, without being tuned to the usage bias of highly expressed genes. We perform a genome-wide evaluation of codon bias for E.coli, comparing CompAI with other widely used indices: tAI, CAI, and Nc. We show that CompAI and tAI capture similar information by being positively correlated with gene conservation, measured by the Evolutionary Retention Index (ERI), and essentiality, whereas, CAI and Nc appear to be less sensitive to evolutionary-functional parameters. Notably, the rate of variation of tAI and CompAI with ERI allows to obtain sets of genes that consistently belong to specific clusters of orthologous genes (COGs). We also investigate the correlation of codon bias at the genomic level with the network features of protein-protein interactions in E.coli. We find that the most densely connected communities of the network share a similar level of codon bias (as measured by CompAI and tAI). Conversely, a small difference in codon bias between two genes is, statistically, a prerequisite for the corresponding proteins to interact. Importantly, among all codon bias indices, CompAI turns out to have the most coherent distribution over the communities of the interactome, pointing to the significance of competition among cognate and near-cognate tRNAs for explaining codon usage adaptation. Notably, CompAI may potentially correlate with translation speed measurements, by accounting for the specific delay induced by wobble-pairing between codons and anticodons. PMID:26566157

  6. Predicting the Binding Patterns of Hub Proteins: A Study Using Yeast Protein Interaction Networks

    PubMed Central

    Andorf, Carson M.; Honavar, Vasant; Sen, Taner Z.

    2013-01-01

    Background Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH) with one or two binding sites, or multiple-interface hubs (MIH) with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations) or party hubs (i.e., simultaneously interact with multiple partners). Methodology Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB) protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques. Conclusions Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions. Availability We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu. PMID:23431393

  7. Microfluidic parallel circuit for measurement of hydraulic resistance.

    PubMed

    Choi, Sungyoung; Lee, Myung Gwon; Park, Je-Kyun

    2010-01-01

    We present a microfluidic parallel circuit that directly compares the test channel of an unknown hydraulic resistance with the reference channel with a known resistance, thereby measuring the unknown resistance without any measurement setup, such as standard pressure gauges. Many of microfluidic applications require the precise transport of fluid along a channel network with complex patterns. Therefore, it is important to accurately characterize and measure the hydraulic resistance of each channel segment, and determines whether the device principle works well. However, there is no fluidic device that includes features, such as the ability to diagnose microfluidic problems by measuring the hydraulic resistance of a microfluidic component in microscales. To address the above need, we demonstrate a simple strategy to measure an unknown hydraulic resistance, by characterizing the hydraulic resistance of microchannels with different widths and defining an equivalent linear channel of a microchannel with repeated patterns of a sudden contraction and expansion.

  8. Flock-based microfluidics.

    PubMed

    Hitzbleck, Martina; Lovchik, Robert D; Delamarche, Emmanuel

    2013-05-21

    Flock-based microfluidics are created by depositing hydrophilic microfibers on an adhesive-coated substrate using an electric field. This enables the fabrication of self-powered microfluidics from one or more different kinds of fibers that form 2D and 3D flowpaths, which can wick 40 microliters of liquid per square centimeter. With this approach, large areas of functional wicking materials can be produced at extremely low cost.

  9. Protein patterns of the Brassica rapa ovules and seeds under altered gravity.

    PubMed

    Sozinov, Igor; Kozub, Natalia; Popova, Antonina

    2004-07-01

    Electrophoretic investigation of protein patterns of Brassica rapa L. ovules and seeds from plants grown under clinorotation and in the laboratory control was carried out. Ovules at different stages (7 and 18 days after pollination) and mature seeds were analyzed. Polymorphism of seed storage proteins of B. rapa was taken into consideration in analysis of changes in ovule protein patterns under clinorotation. The appearance of a protein component in the region of about 43 kDa was detected in protein patterns of 7-day-old and 18-day-old ovules in the clinostat variants. Under altered gravity, in 18-day-old ovules, the appearance of a protein in the region of about 70 kDa was also revealed. The appearance of the protein component with the similar mobility (about 43 kDa) in ovules of different age from plants grown at clinorotation suggests that synthesis of this protein may be associated with the plant response to altered gravity. However, the investigation of the nature of this protein and its role requires further research to rule out its appearance because of genotypic differences between ovules of the control and experimental variants.

  10. MEMS in microfluidic channels.

    SciTech Connect

    Ashby, Carol Iris Hill; Okandan, Murat; Michalske, Terry A.; Sounart, Thomas L.; Matzke, Carolyn M.

    2004-03-01

    Microelectromechanical systems (MEMS) comprise a new class of devices that include various forms of sensors and actuators. Recent studies have shown that microscale cantilever structures are able to detect a wide range of chemicals, biomolecules or even single bacterial cells. In this approach, cantilever deflection replaces optical fluorescence detection thereby eliminating complex chemical tagging steps that are difficult to achieve with chip-based architectures. A key challenge to utilizing this new detection scheme is the incorporation of functionalized MEMS structures within complex microfluidic channel architectures. The ability to accomplish this integration is currently limited by the processing approaches used to seal lids on pre-etched microfluidic channels. This report describes Sandia's first construction of MEMS instrumented microfluidic chips, which were fabricated by combining our leading capabilities in MEMS processing with our low-temperature photolithographic method for fabricating microfluidic channels. We have explored in-situ cantilevers and other similar passive MEMS devices as a new approach to directly sense fluid transport, and have successfully monitored local flow rates and viscosities within microfluidic channels. Actuated MEMS structures have also been incorporated into microfluidic channels, and the electrical requirements for actuation in liquids have been quantified with an elegant theory. Electrostatic actuation in water has been accomplished, and a novel technique for monitoring local electrical conductivities has been invented.

  11. Highly efficient and selective isolation of rare tumor cells using a microfluidic chip with wavy-herringbone micro-patterned surfaces.

    PubMed

    Wang, Shunqiang; Thomas, Antony; Lee, Elaine; Yang, Shu; Cheng, Xuanhong; Liu, Yaling

    2016-04-01

    Circulating tumor cells (CTCs) in peripheral blood have been recognized as a general biomarker for diagnosing cancer and providing guidance for personalized treatments. Yet due to their rarity, the challenge for their clinical utility lies in the efficient isolation while avoiding the capture of other non-targeted white blood cells (WBCs). In this paper, a wavy-herringbone (HB) microfluidic chip coated with antibody directly against epithelial cell adhesion molecule (anti-EpCAM) was developed for highly efficient and selective isolation of tumor cells from tumor cell-spiked whole blood samples. By extending the concept of the hallmark HB-Chip in the literature, the wavy-HB chip not only achieves high capture efficiency (up to 85.0%) by micro-vortexes induced by HB structures, but also achieves high purity (up to 39.4%) due to the smooth wavy microstructures. These smooth wavy-HB structures eliminate the ultra-low shear rate regions in the traditional grooved-HB structures that lead to non-specific trapping of cells. Compared with the grooved-HB chip with sharp corners, the wavy-HB chip shows significantly higher purity while maintaining similarly high capture efficiency. Furthermore, the wavy-HB chip has up to 11% higher captured cell viability over the grooved-HB chip. The distributions of tumor cells and WBCs along the grooves and waves are investigated to help understand the mechanisms behind the better performance of the wavy-HB chip. The wavy-HB chip may serve as a promising platform for CTC capture and cancer diagnosis. PMID:26907962

  12. Highly efficient and selective isolation of rare tumor cells using a microfluidic chip with wavy-herringbone micro-patterned surfaces.

    PubMed

    Wang, Shunqiang; Thomas, Antony; Lee, Elaine; Yang, Shu; Cheng, Xuanhong; Liu, Yaling

    2016-04-01

    Circulating tumor cells (CTCs) in peripheral blood have been recognized as a general biomarker for diagnosing cancer and providing guidance for personalized treatments. Yet due to their rarity, the challenge for their clinical utility lies in the efficient isolation while avoiding the capture of other non-targeted white blood cells (WBCs). In this paper, a wavy-herringbone (HB) microfluidic chip coated with antibody directly against epithelial cell adhesion molecule (anti-EpCAM) was developed for highly efficient and selective isolation of tumor cells from tumor cell-spiked whole blood samples. By extending the concept of the hallmark HB-Chip in the literature, the wavy-HB chip not only achieves high capture efficiency (up to 85.0%) by micro-vortexes induced by HB structures, but also achieves high purity (up to 39.4%) due to the smooth wavy microstructures. These smooth wavy-HB structures eliminate the ultra-low shear rate regions in the traditional grooved-HB structures that lead to non-specific trapping of cells. Compared with the grooved-HB chip with sharp corners, the wavy-HB chip shows significantly higher purity while maintaining similarly high capture efficiency. Furthermore, the wavy-HB chip has up to 11% higher captured cell viability over the grooved-HB chip. The distributions of tumor cells and WBCs along the grooves and waves are investigated to help understand the mechanisms behind the better performance of the wavy-HB chip. The wavy-HB chip may serve as a promising platform for CTC capture and cancer diagnosis.

  13. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein.

    PubMed

    Buffalo, Cosmo Z; Bahn-Suh, Adrian J; Hirakis, Sophia P; Biswas, Tapan; Amaro, Rommie E; Nizet, Victor; Ghosh, Partho

    2016-01-01

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ∼90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant 'reading head' in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M-C4BP interaction, and also inform a path towards vaccine design. PMID:27595425

  14. Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development

    PubMed Central

    Stanic, Karen; Saldivia, Natalia; Förstera, Benjamín; Torrejón, Marcela; Montecinos, Hernán; Caprile, Teresa

    2016-01-01

    Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons. PMID:27733818

  15. Adhesion of adipose-derived mesenchymal stem cells to glycosaminoglycan surfaces with different protein patterns.

    PubMed

    Soares da Costa, Diana; Márquez-Posadas, Maria del Carmen; Araujo, Ana R; Yang, Yuan; Merino, Santos; Groth, Thomas; Reis, Rui L; Pashkuleva, Iva

    2015-05-13

    Proteins and glycosaminoglycans (GAGs) are the main constituents of the extracellular matrix (ECM). They act in synergism and are equally critical for the development, growth, function, or survival of an organism. In this work, we developed surfaces that display these two classes of biomacromolecules, namely, GAGs and proteins, in a spatially controlled fashion. The generated surfaces can be used as a minimalistic but straightforward model aiding the elucidation of cell-ECM interactions. GAGs (hyaluronic acid and heparin) were covalently bound to amino functionalized surfaces, and albumin or fibronectin was patterned by microcontact printing on top of them. We demonstrate that adipose-derived stem cells (ASCs) can adhere either on the protein or on the GAG pattern as a function of the patterned molecules. ASCs found on the GAG pattern had different morphology and expressed different surface markers than the cells adhered on the protein pattern. ASCs morphology and spreading were also dependent on the size of the pattern. These results show that the developed supports can also be used for ASCs differentiation into different lineages.

  16. Research highlights: microfluidics meets big data.

    PubMed

    Tseng, Peter; Weaver, Westbrook M; Masaeli, Mahdokht; Owsley, Keegan; Di Carlo, Dino

    2014-03-01

    In this issue we highlight a collection of recent work in which microfluidic parallelization and automation have been employed to address the increasing need for large amounts of quantitative data concerning cellular function--from correlating microRNA levels to protein expression, increasing the throughput and reducing the noise when studying protein dynamics in single-cells, and understanding how signal dynamics encodes information. The painstaking dissection of cellular pathways one protein at a time appears to be coming to an end, leading to more rapid discoveries which will inevitably translate to better cellular control--in producing useful gene products and treating disease at the individual cell level. From these studies it is also clear that development of large scale mutant or fusion libraries, automation of microscopy, image analysis, and data extraction will be key components as microfluidics contributes its strengths to aid systems biology moving forward.

  17. An ultra-sensitive microfluidic immunoassay using living radical polymerization and porous polymer monoliths.

    SciTech Connect

    Abhyankar, Vinay V.; Singh, Anup K.; Hatch, Anson V.

    2010-07-01

    We present a platform that combines patterned photopolymerized polymer monoliths with living radical polymerization (LRP) to develop a low cost microfluidic based immunoassay capable of sensitive (low to sub pM) and rapid (<30 minute) detection of protein in 100 {micro}L sample. The introduction of LRP functionality to the porous monolith allows one step grafting of functionalized affinity probes from the monolith surface while the composition of the hydrophilic graft chain reduces non-specific interactions and helps to significantly improve the limit of detection.

  18. Punch card programmable microfluidics.

    PubMed

    Korir, George; Prakash, Manu

    2015-01-01

    Small volume fluid handling in single and multiphase microfluidics provides a promising strategy for efficient bio-chemical assays, low-cost point-of-care diagnostics and new approaches to scientific discoveries. However multiple barriers exist towards low-cost field deployment of programmable microfluidics. Incorporating multiple pumps, mixers and discrete valve based control of nanoliter fluids and droplets in an integrated, programmable manner without additional required external components has remained elusive. Combining the idea of punch card programming with arbitrary fluid control, here we describe a self-contained, hand-crank powered, multiplex and robust programmable microfluidic platform. A paper tape encodes information as a series of punched holes. A mechanical reader/actuator reads these paper tapes and correspondingly executes operations onto a microfluidic chip coupled to the platform in a plug-and-play fashion. Enabled by the complexity of codes that can be represented by a series of holes in punched paper tapes, we demonstrate independent control of 15 on-chip pumps with enhanced mixing, normally-closed valves and a novel on-demand impact-based droplet generator. We demonstrate robustness of operation by encoding a string of characters representing the word "PUNCHCARD MICROFLUIDICS" using the droplet generator. Multiplexing is demonstrated by implementing an example colorimetric water quality assays for pH, ammonia, nitrite and nitrate content in different water samples. With its portable and robust design, low cost and ease-of-use, we envision punch card programmable microfluidics will bring complex control of microfluidic chips into field-based applications in low-resource settings and in the hands of children around the world. PMID:25738834

  19. Punch card programmable microfluidics.

    PubMed

    Korir, George; Prakash, Manu

    2015-01-01

    Small volume fluid handling in single and multiphase microfluidics provides a promising strategy for efficient bio-chemical assays, low-cost point-of-care diagnostics and new approaches to scientific discoveries. However multiple barriers exist towards low-cost field deployment of programmable microfluidics. Incorporating multiple pumps, mixers and discrete valve based control of nanoliter fluids and droplets in an integrated, programmable manner without additional required external components has remained elusive. Combining the idea of punch card programming with arbitrary fluid control, here we describe a self-contained, hand-crank powered, multiplex and robust programmable microfluidic platform. A paper tape encodes information as a series of punched holes. A mechanical reader/actuator reads these paper tapes and correspondingly executes operations onto a microfluidic chip coupled to the platform in a plug-and-play fashion. Enabled by the complexity of codes that can be represented by a series of holes in punched paper tapes, we demonstrate independent control of 15 on-chip pumps with enhanced mixing, normally-closed valves and a novel on-demand impact-based droplet generator. We demonstrate robustness of operation by encoding a string of characters representing the word "PUNCHCARD MICROFLUIDICS" using the droplet generator. Multiplexing is demonstrated by implementing an example colorimetric water quality assays for pH, ammonia, nitrite and nitrate content in different water samples. With its portable and robust design, low cost and ease-of-use, we envision punch card programmable microfluidics will bring complex control of microfluidic chips into field-based applications in low-resource settings and in the hands of children around the world.

  20. Integrated microfluidic probe station

    NASA Astrophysics Data System (ADS)

    Perrault, C. M.; Qasaimeh, M. A.; Brastaviceanu, T.; Anderson, K.; Kabakibo, Y.; Juncker, D.

    2010-11-01

    The microfluidic probe (MFP) consists of a flat, blunt tip with two apertures for the injection and reaspiration of a microjet into a solution—thus hydrodynamically confining the microjet—and is operated atop an inverted microscope that enables live imaging. By scanning across a surface, the microjet can be used for surface processing with the capability of both depositing and removing material; as it operates under immersed conditions, sensitive biological materials and living cells can be processed. During scanning, the MFP is kept immobile and centered over the objective of the inverted microscope, a few micrometers above a substrate that is displaced by moving the microscope stage and that is flushed continuously with the microjet. For consistent and reproducible surface processing, the gap between the MFP and the substrate, the MFP's alignment, the scanning speed, the injection and aspiration flow rates, and the image capture need all to be controlled and synchronized. Here, we present an automated MFP station that integrates all of these functionalities and automates the key operational parameters. A custom software program is used to control an independent motorized Z stage for adjusting the gap, a motorized microscope stage for scanning the substrate, up to 16 syringe pumps for injecting and aspirating fluids, and an inverted fluorescence microscope equipped with a charge-coupled device camera. The parallelism between the MFP and the substrate is adjusted using manual goniometer at the beginning of the experiment. The alignment of the injection and aspiration apertures along the scanning axis is performed using a newly designed MFP screw holder. We illustrate the integrated MFP station by the programmed, automated patterning of fluorescently labeled biotin on a streptavidin-coated surface.

  1. Regular Patterns for Proteome-Wide Distribution of Protein Abundance across Species

    PubMed Central

    Jiang, Ying; Ying, Wantao; Wu, Songfeng; Zhu, Yunping; Liu, Siqi; Yang, Pengyuan; Qian, Xiaohong; He, Fuchu

    2012-01-01

    A proteome of the bio-entity, including cell, tissue, organ, and organism, consists of proteins of diverse abundance. The principle that determines the abundance of different proteins in a proteome is of fundamental significance for an understanding of the building blocks of the bio-entity. Here, we report three regular patterns in the proteome-wide distribution of protein abundance across species such as human, mouse, fly, worm, yeast, and bacteria: in most cases, protein abundance is positively correlated with the protein's origination time or sequence conservation during evolution; it is negatively correlated with the protein's domain number and positively correlated with domain coverage in protein structure, and the correlations became stronger during the course of evolution; protein abundance can be further stratified by the function of the protein, whereby proteins that act on material conversion and transportation (mass category) are more abundant than those that act on information modulation (information category). Thus, protein abundance is intrinsically related to the protein's inherent characters of evolution, structure, and function. PMID:22427835

  2. Protein Adsorption on Patterned Hydroxyapatite Thin Films Fabricated by Pulsed Laser Deposition

    NASA Astrophysics Data System (ADS)

    Kusunoki, Masanobu; Kawasima, Masami; Nishikawa, Hiroaki; Morimoto, Koichi; Hayami, Takashi; Hontsu, Shigeki; Kawai, Tomoji

    2005-02-01

    Protein adsorption on hydroxyapatite (HAP) thin film was investigated before and after patterning. Hydroxyapatite thin film 100 nm thick was deposited by pulsed laser deposition. The film was patterned by photolithography and wet etching with HCl solution. Proteins (phospholyrase b, bovine serum albumin, and others) labeled with fluorescein isothiocyanate (FITC) were used as the reagent. After the HAP film was soaked in the reagent and washed with pure water, a conspicuous contrast in FITC was observed between the HAP pattern and the glass substrate (or photoresist). This behavior showed that the biocompatibility of the HAP thin film was not influenced by the patterning process. Our technique for HAP thin film is adaptable for applications involving biosensors as electronic devices and scaffolds for tissue culture.

  3. Changes in protein patterns and in vivo protein synthesis during senescence of hibiscus petals. [Hibiscus rosa-sinensis

    SciTech Connect

    Woodson, W.R.; Handa, A.K.

    1986-04-01

    Changes in proteins associated with senescence of the flowers of Hibiscus rosa-sinensis was studied using SDS-PAGE. Total extractable protein from petals decreased with senescence. Changes were noted in patterns of proteins from aging petals. Flower opening and senescence was associated with appearance and disappearance of several polypeptides. One new polypeptide with an apparent mw of 41 kd was first seen the day of flower opening and increased to over 9% of the total protein content of senescent petal tissue. Protein synthesis during aging was investigated by following uptake and incorporation of /sup 3/H-leucine into TCA-insoluble fraction of petal discs. Protein synthesis, as evidenced by the percent of label incorporated into the TCA-insoluble fraction, was greatest (32%) the day before flower opening. Senescent petal tissue incorporated 4% of label taken up into protein. Proteins were separated by SDS-PAGE and labelled polypeptides identified by fluorography. In presenescent petal tissue, radioactivity was distributed among several major polypeptides. In senescent tissue, much of the radioactivity was concentrated in the 41 kd polypeptide.

  4. Tuning Fluidic Resistance via Liquid Crystal Microfluidics

    PubMed Central

    Sengupta, Anupam

    2013-01-01

    Flow of molecularly ordered fluids, like liquid crystals, is inherently coupled with the average local orientation of the molecules, or the director. The anisotropic coupling—typically absent in isotropic fluids—bestows unique functionalities to the flowing matrix. In this work, we harness this anisotropy to pattern different pathways to tunable fluidic resistance within microfluidic devices. We use a nematic liquid crystalline material flowing in microchannels to demonstrate passive and active modulation of the flow resistance. While appropriate surface anchoring conditions—which imprint distinct fluidic resistances within microchannels under similar hydrodynamic parameters—act as passive cues, an external field, e.g., temperature, is used to actively modulate the flow resistance in the microfluidic device. We apply this simple concept to fabricate basic fluidic circuits, which can be hierarchically extended to create complex resistance networks, without any additional design or morphological patterning of the microchannels. PMID:24256819

  5. Living anionic polymerization using a microfluidic reactor

    SciTech Connect

    Iida, Kazunori; Chastek, Thomas Q.; Beers, Kathryn L.; Cavicchi, Kevin A.; Chun, Jaehun; Fasolka, Michael J.

    2009-02-01

    Living anionic polymerizations were conducted within aluminum-polyimide microfluidic devices. Polymerizations of styrene in cyclohexane were carried out at various conditions, including elevated temperature (60 °C) and high monomer concentration (42%, by volume). The reactions were safely maintained at a controlled temperature at all points in the reactor. Conducting these reactions in a batch reactor results in uncontrolled heat generation with potentially dangerous rises in pressure. Moreover, the microfluidic nature of these devices allows for flexible 2D designing of the flow channel. Four flow designs were examined (straight, periodically pinched, obtuse zigzag, and acute zigzag channels). The ability to use the channel pattern to increase the level of mixing throughout the reactor was evaluated. When moderately high molecular mass polymers with increased viscosity were made, the patterned channels produced polymers with narrower PDI, indicating that passive mixing arising from the channel design is improving the reaction conditions.

  6. QuasiMotiFinder: protein annotation by searching for evolutionarily conserved motif-like patterns.

    PubMed

    Gutman, Roee; Berezin, Carine; Wollman, Roy; Rosenberg, Yossi; Ben-Tal, Nir

    2005-07-01

    Sequence signature databases such as PROSITE, which include amino acid segments that are indicative of a protein's function, are useful for protein annotation. Lamentably, the annotation is not always accurate. A signature may be falsely detected in a protein that does not carry out the associated function (false positive prediction, FP) or may be overlooked in a protein that does carry out the function (false negative prediction, FN). A new approach has emerged in which a signature is replaced with a sequence profile, calculated based on multiple sequence alignment (MSA) of homologous proteins that share the same function. This approach, which is superior to the simple pattern search, essentially searches with the sequence of the query protein against an MSA library. We suggest here an alternative approach, implemented in the QuasiMotiFinder web server (http://quasimotifinder.tau.ac.il/), which is based on a search with an MSA of homologous query proteins against the original PROSITE signatures. The explicit use of the average evolutionary conservation of the signature in the query proteins significantly reduces the rate of FP prediction compared with the simple pattern search. QuasiMotiFinder also has a reduced rate of FN prediction compared with simple pattern searches, since the traditional search for precise signatures has been replaced by a permissive search for signature-like patterns that are physicochemically similar to known signatures. Overall, QuasiMotiFinder and the profile search are comparable to each other in terms of performance. They are also complementary to each other in that signatures that are falsely detected in (or overlooked by) one may be correctly detected by the other.

  7. Characterization of a nanoscale S-layer protein based template for biomolecular patterning.

    PubMed

    Wong, Wing Sze; Yung, Pun To

    2014-01-01

    Well organized template for biomolecular conjugation is the foundation for biosensing. Most of the current devices are fabricated using lithographic patterning processes and self-assembly monolayer (SAM) methods. However, the research toward developing a sub-10 nm patterned, self-regenerated template on various types of substrates is limited, mainly due to the limited functional groups of the building material. Bacterial surface layer proteins (S-layer proteins) can self-assemble into ordered lattice with regular pore sizes of 2-8 nm on different material supports and interfaces. The ordered structure can regenerate after extreme variations of solvent conditions. In this work, we developed a nanoscale biomolecular template based on S-layer proteins on gold surface for fabrication of sensing layer in biosensors. S-layer proteins were isolated from Bacillus cereus, Lysinibacillus sphaericus and Geobacillus stearothermophilus. Protein concentrations were measured by Bradford assay. The protein purities were verified by SDS-PAGE, showing molecular weights ranging from 97-135 kDa. The hydrophilicity of the substrate surface was measured after surface treatments of protein recrystallization. Atomic force microscopic (AFM) measurement was performed on substrate surface, indicating a successful immobilization of a monolayer of S-layer protein with 8-9 nm height on gold surface. The template can be applied on various material supports and acts as a self-regenerated sensing layer of biosensors in the future. PMID:25570568

  8. Collective waves in dense and confined microfluidic droplet arrays.

    PubMed

    Schiller, Ulf D; Fleury, Jean-Baptiste; Seemann, Ralf; Gompper, Gerhard

    2015-08-01

    Excitation mechanisms for collective waves in confined dense one-dimensional microfluidic droplet arrays are investigated by experiments and computer simulations. We demonstrate that distinct modes can be excited by creating specific 'defect' patterns in flowing droplet trains. Excited longitudinal modes exhibit a short-lived cascade of pairs of laterally displacing droplets. Transversely excited modes obey the dispersion relation of microfluidic phonons and induce a coupling between longitudinal and transverse modes, whose origin is the hydrodynamic interaction of the droplets with the confining walls. Moreover, we investigate the long-time behaviour of the oscillations and discuss possible mechanisms for the onset of instabilities. Our findings demonstrate that the collective dynamics of microfluidic droplet ensembles can be studied particularly well in dense and confined systems. Experimentally, the ability to control microfluidic droplets may allow the modulation of the refractive index of optofluidic crystals, which is a promising approach for the production of dynamically programmable metamaterials. PMID:26107262

  9. Rapid prototyping of multiphase microfluidics with robotic cutters

    NASA Astrophysics Data System (ADS)

    Li, Zidong; Zhao, Zhengtuo; Lo, Joe Fu-jiou

    2014-03-01

    Microfluidic devices offer novel techniques to address biological and biomedical issues. Standard microfluidic fabrication uses photolithography to pattern channels on silicon wafers with high resolution. Even the relatively straightforward SU8 and soft lithography in microfluidics require investing and training in photolithography, which is also time consuming due to complicated thick resist procedures, including sensitive substrate pretreatment, coating, soft bake, expose, post-exposure bake, and developing steps. However, for applications where low resolution (>200 μm) and high turn-around (> 4 designs/day) prototyping are met with little or no lithography infrastructure, robotic cutters [1] offer flexible options for making glass and PDMS microfluidics. We describe the use of robotics cutters for designing microfluidic geometries, and compliment it with safe glass etching, with depths down to 60 μm. Soft lithography patterning of 200 μm thick PDMS membrane was also explored. Without high equipment investment and lengthy student training, both glass and PDMS microfluidics can be achieved in small facilities using this technique.

  10. Microfluidic Mixing: A Review

    PubMed Central

    Lee, Chia-Yen; Chang, Chin-Lung; Wang, Yao-Nan; Fu, Lung-Ming

    2011-01-01

    The aim of microfluidic mixing is to achieve a thorough and rapid mixing of multiple samples in microscale devices. In such devices, sample mixing is essentially achieved by enhancing the diffusion effect between the different species flows. Broadly speaking, microfluidic mixing schemes can be categorized as either “active”, where an external energy force is applied to perturb the sample species, or “passive”, where the contact area and contact time of the species samples are increased through specially-designed microchannel configurations. Many mixers have been proposed to facilitate this task over the past 10 years. Accordingly, this paper commences by providing a high level overview of the field of microfluidic mixing devices before describing some of the more significant proposals for active and passive mixers. PMID:21686184

  11. Microfluidic platforms for mechanobiology

    PubMed Central

    Polacheck, William J.; Li, Ran; Uzel, Sebastien G. M.

    2013-01-01

    Mechanotransduction has been a topic of considerable interest since early studies demonstrated a link between mechanical force and biological response. Until recently, studies of fundamental phenomena were based either on in vivo experiments with limited control or direct access, or on large-scale in vitro studies lacking many of the potentially important physiological factors. With the advent of microfluidics, many of the previous limitations of in vitro testing were eliminated or reduced through greater control or combined functionalities. At the same time, imaging capabilities were tremendously enhanced. In this review, we discuss how microfluidics has transformed the study of mechanotransduction. This is done in the context of the various cell types that exhibit force-induced responses and the new biological insights that have been elucidated. We also discuss new microfluidic studies that could produce even more realistic models of in vivo conditions by combining multiple stimuli or creating a more realistic microenvironment. PMID:23649165

  12. The kinase regulator mob1 acts as a patterning protein for stentor morphogenesis.

    PubMed

    Slabodnick, Mark M; Ruby, J Graham; Dunn, Joshua G; Feldman, Jessica L; DeRisi, Joseph L; Marshall, Wallace F

    2014-05-01

    Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1--with conserved functions in cell division and morphogenesis from plants to humans-as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system. PMID:24823688

  13. The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis

    PubMed Central

    Slabodnick, Mark M.; Ruby, J. Graham; Dunn, Joshua G.; Feldman, Jessica L.; DeRisi, Joseph L.; Marshall, Wallace F.

    2014-01-01

    Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1—with conserved functions in cell division and morphogenesis from plants to humans—as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system. PMID:24823688

  14. Punch Card Programmable Microfluidics

    PubMed Central

    Korir, George; Prakash, Manu

    2015-01-01

    Small volume fluid handling in single and multiphase microfluidics provides a promising strategy for efficient bio-chemical assays, low-cost point-of-care diagnostics and new approaches to scientific discoveries. However multiple barriers exist towards low-cost field deployment of programmable microfluidics. Incorporating multiple pumps, mixers and discrete valve based control of nanoliter fluids and droplets in an integrated, programmable manner without additional required external components has remained elusive. Combining the idea of punch card programming with arbitrary fluid control, here we describe a self-contained, hand-crank powered, multiplex and robust programmable microfluidic platform. A paper tape encodes information as a series of punched holes. A mechanical reader/actuator reads these paper tapes and correspondingly executes operations onto a microfluidic chip coupled to the platform in a plug-and-play fashion. Enabled by the complexity of codes that can be represented by a series of holes in punched paper tapes, we demonstrate independent control of 15 on-chip pumps with enhanced mixing, normally-closed valves and a novel on-demand impact-based droplet generator. We demonstrate robustness of operation by encoding a string of characters representing the word “PUNCHCARD MICROFLUIDICS” using the droplet generator. Multiplexing is demonstrated by implementing an example colorimetric water quality assays for pH, ammonia, nitrite and nitrate content in different water samples. With its portable and robust design, low cost and ease-of-use, we envision punch card programmable microfluidics will bring complex control of microfluidic chips into field-based applications in low-resource settings and in the hands of children around the world. PMID:25738834

  15. Development & Characterization of Multifunctional Microfluidic Materials

    NASA Astrophysics Data System (ADS)

    Ucar, Ahmet Burak

    developing 'smart' windows and heat management. To better design new color changing elastomers, we investigated the role of the network geometry on liquid replacement efficiency with the aid of a multiphysics modeling and simulation software package, COMSOL. We simulated the liquid flow in various network geometries. Serpentine, parallel channel and lattice networks, as well as their tapered versions were compared. The comparison criteria were based on rapid and uniform liquid replacement with the least amount of dye/liquid required, for which we set multiple constraints such as constant inlet pressure or total channel area. We demonstrated that the tapered lattice type network provided the most rapid and uniform replacement with minimal liquid waste. Next, we designed a simple and inexpensive liquid dispensing microfluidic material which does not require complex micromachining techniques or automated actuators. It consisted of only a PDMS matrix with embedded chambers and channels. 'Pores/slits' were made on the surface and the liquid was released by contact on the dispensing surface of the material. We varied the network design, geometry, dimension, slit shape and length, and tested the material's liquid release performance. Promising preliminary results were obtained but for an end product with repeatable and reproducible performance, both material fabrication and characterization need to be improved further. Finally, we describe an alternative material/method for the fabrication of microfluidic materials. We aimed to replace the conventional fabrication material PDMS with Polyethylene (PE) sheets. The sheets were as transparent and flexible as PDMS, and also thinner. Channel patterns were drawn with a polymer solution of PolyVinylAlcohol (PVA), which is immiscible with PE, and captured in between the two PE sheets. After fusing the PE sheets on a hot press, PVA was washed off with water, so that the 'microfluidic channels' were successfully created. The produced channel

  16. Development & Characterization of Multifunctional Microfluidic Materials

    NASA Astrophysics Data System (ADS)

    Ucar, Ahmet Burak

    developing 'smart' windows and heat management. To better design new color changing elastomers, we investigated the role of the network geometry on liquid replacement efficiency with the aid of a multiphysics modeling and simulation software package, COMSOL. We simulated the liquid flow in various network geometries. Serpentine, parallel channel and lattice networks, as well as their tapered versions were compared. The comparison criteria were based on rapid and uniform liquid replacement with the least amount of dye/liquid required, for which we set multiple constraints such as constant inlet pressure or total channel area. We demonstrated that the tapered lattice type network provided the most rapid and uniform replacement with minimal liquid waste. Next, we designed a simple and inexpensive liquid dispensing microfluidic material which does not require complex micromachining techniques or automated actuators. It consisted of only a PDMS matrix with embedded chambers and channels. 'Pores/slits' were made on the surface and the liquid was released by contact on the dispensing surface of the material. We varied the network design, geometry, dimension, slit shape and length, and tested the material's liquid release performance. Promising preliminary results were obtained but for an end product with repeatable and reproducible performance, both material fabrication and characterization need to be improved further. Finally, we describe an alternative material/method for the fabrication of microfluidic materials. We aimed to replace the conventional fabrication material PDMS with Polyethylene (PE) sheets. The sheets were as transparent and flexible as PDMS, and also thinner. Channel patterns were drawn with a polymer solution of PolyVinylAlcohol (PVA), which is immiscible with PE, and captured in between the two PE sheets. After fusing the PE sheets on a hot press, PVA was washed off with water, so that the 'microfluidic channels' were successfully created. The produced channel

  17. A programmable and reconfigurable microfluidic chip.

    PubMed

    Renaudot, Raphael; Agache, Vincent; Fouillet, Yves; Laffite, Guillaume; Bisceglia, Emilie; Jalabert, Laurent; Kumemura, Momoko; Collard, Dominique; Fujita, Hiroyuki

    2013-12-01

    This article reports an original concept enabling the rapid fabrication of continuous-flow microfluidic chips with a programmable and reconfigurable geometry. The concept is based on a digital microfluidic platform featuring an array of individually addressable electrodes. A selection of electrodes is switched on sequentially to create a de-ionized (DI) water finger specific pattern, while the surrounding medium consists of liquid-phase paraffin. The water displacement is induced by both electrowetting on dielectric and liquid dielectrophoresis phenomena. Once the targeted DI water pattern is obtained, the chip temperature is lowered by turning on an integrated thermoelectric cooler, forming channel structures made of solidified paraffin with edges delimitated by the DI water pattern. As a result, the chip can be used afterwards to conduct in-flow continuous microfluidic experiments. This process is resettable and reversible by heating up the chip to melt the paraffin and reconfigure the microchannel design on demand, offering the advantages of cost, adaptability, and robustness. This paper reports experimental results describing the overall concept, which is illustrated with typical and basic fluidic geometries.

  18. In vitro evaluation and in vivo demonstration of a biomimetic, hemocompatible, microfluidic artificial lung.

    PubMed

    Kovach, K M; LaBarbera, M A; Moyer, M C; Cmolik, B L; van Lunteren, E; Sen Gupta, A; Capadona, J R; Potkay, J A

    2015-03-01

    Despite the promising potential of microfluidic artificial lungs, current designs suffer from short functional lifetimes due to surface chemistry and blood flow patterns that act to reduce hemocompatibility. Here, we present the first microfluidic artificial lung featuring a hemocompatible surface coating and a biomimetic blood path. The polyethylene-glycol (PEG) coated microfluidic lung exhibited a significantly improved in vitro lifetime compared to uncoated controls as well as consistent and significantly improved gas exchange over the entire testing period. Enabled by our hemocompatible PEG coating, we additionally describe the first extended (3 h) in vivo demonstration of a microfluidic artificial lung.

  19. Microfluidic Flame Barrier

    NASA Technical Reports Server (NTRS)

    Mungas, Gregory S. (Inventor); Fisher, David J. (Inventor); Mungas, Christopher (Inventor)

    2013-01-01

    Propellants flow through specialized mechanical hardware that is designed for effective and safe ignition and sustained combustion of the propellants. By integrating a micro-fluidic porous media element between a propellant feed source and the combustion chamber, an effective and reliable propellant injector head may be implemented that is capable of withstanding transient combustion and detonation waves that commonly occur during an ignition event. The micro-fluidic porous media element is of specified porosity or porosity gradient selected to be appropriate for a given propellant. Additionally the propellant injector head design integrates a spark ignition mechanism that withstands extremely hot running conditions without noticeable spark mechanism degradation.

  20. Experimental Microfluidic System

    NASA Technical Reports Server (NTRS)

    Culbertson, Christopher; Gonda, Steve; Ramsey, John Michael

    2005-01-01

    The ultimate goal of this project is to integrate microfluidic devices with NASA's space bioreactor systems. In such a system, the microfluidic device would provide realtime feedback control of the bioreactor by monitoring pH, glucose, and lactate levels in the cell media; and would provide an analytical capability to the bioreactor in exterrestrial environments for monitoring bioengineered cell products and health changes in cells due to environmental stressors. Such integrated systems could be used as biosentinels both in space and on planet surfaces. The objective is to demonstrate the ability of microfabricated devices to repeatedly and reproducibly perform bead cytometry experiments in micro, lunar, martian, and hypergravity (1.8g).

  1. Inkjet printing of UV-curable adhesive and dielectric inks for microfluidic devices.

    PubMed

    Hamad, E M; Bilatto, S E R; Adly, N Y; Correa, D S; Wolfrum, B; Schöning, M J; Offenhäusser, A; Yakushenko, A

    2016-01-01

    Bonding of polymer-based microfluidics to polymer substrates still poses a challenge for Lab-On-a-Chip applications. Especially, when sensing elements are incorporated, patterned deposition of adhesives with curing at ambient conditions is required. Here, we demonstrate a fabrication method for fully printed microfluidic systems with sensing elements using inkjet and stereolithographic 3D-printing.

  2. Laser trapping and patterning of protein microcrystals: Toward highly integrated protein microarrays

    NASA Astrophysics Data System (ADS)

    Hosokawa, Yoichiroh; Matsumura, Satoshi; Masuhara, Hiroshi; Ikeda, Keiko; Shimo-oka, Ai; Mori, Hajime

    2004-09-01

    Some insect virus infections occlude into a crystalline matrix consisting of a protein named polyhedrin. The shape of the matrix is a cubic polyhedron of the size of a few micrometers. Recently it was shown that these polyhedra could immobilize various functional proteins within them. Therefore, the polyhedron is interesting as an element in a protein chip. In this work, individual polyhedra were arrayed and bonded under a microscope by focused laser beams, with the aim of fabricating a highly integrated protein chip. The polyhedron was trapped and transferred to a suitable position on a polymer substrate by optical trapping with a 1064nmNd3+:YAG (YAG, yttrium aluminum garnet) laser. To bond the polyhedron on the substrate, the polymer surface was mechanically and chemically modified by multiphoton absorption of a 120fs, 800nm femtosecond Ti: sapphire laser, which results in strong adhesion of the polyhedron to the substrate. The arraying and bonding of polyhedra were successful, to a precision of about 1μm, with this procedure. The biological activity of polyhedra after these laser irradiations was confirmed by the fluorescence of green fluorescent protein occluded in the polyhedrin matrix.

  3. Constitutive patterns of gene expression regulated by RNA-binding proteins

    PubMed Central

    2014-01-01

    Background RNA-binding proteins regulate a number of cellular processes, including synthesis, folding, translocation, assembly and clearance of RNAs. Recent studies have reported that an unexpectedly large number of proteins are able to interact with RNA, but the partners of many RNA-binding proteins are still uncharacterized. Results We combined prediction of ribonucleoprotein interactions, based on catRAPID calculations, with analysis of protein and RNA expression profiles from human tissues. We found strong interaction propensities for both positively and negatively correlated expression patterns. Our integration of in silico and ex vivo data unraveled two major types of protein–RNA interactions, with positively correlated patterns related to cell cycle control and negatively correlated patterns related to survival, growth and differentiation. To facilitate the investigation of protein–RNA interactions and expression networks, we developed the catRAPID express web server. Conclusions Our analysis sheds light on the role of RNA-binding proteins in regulating proliferation and differentiation processes, and we provide a data exploration tool to aid future experimental studies. PMID:24401680

  4. SplitPocket: identification of protein functional surfaces and characterization of their spatial patterns.

    PubMed

    Tseng, Yan Yuan; Dupree, Craig; Chen, Z Jeffrey; Li, Wen-Hsiung

    2009-07-01

    SplitPocket (http://pocket.uchicago.edu/) is a web server to identify functional surfaces of protein from structure coordinates. Using the Alpha Shape Theory, we previously developed an analytical approach to identify protein functional surfaces by the geometric concept of a split pocket, which is a pocket split by a binding ligand. Our geometric approach extracts site-specific spatial information from coordinates of structures. To reduce the search space, probe radii are designed according to the physicochemical textures of molecules. The method uses the weighted Delaunay triangulation and the discrete flow algorithm to obtain geometric measurements and spatial patterns for each predicted pocket. It can also measure the hydrophobicity on a surface patch. Furthermore, we quantify the evolutionary conservation of surface patches by an index derived from the entropy scores in HSSP (homology-derived secondary structure of proteins). We have used the method to examine approximately 1.16 million potential pockets and identified the split pockets in >26,000 structures in the Protein Data Bank. This integrated web server of functional surfaces provides a source of spatial patterns to serve as templates for predicting the functional surfaces of unbound structures involved in binding activities. These spatial patterns should also be useful for protein functional inference, structural evolution and drug design.

  5. Distinct expression patterns of ICK/MAK/MOK protein kinases in the intestine implicate functional diversity.

    PubMed

    Chen, Tufeng; Wu, Di; Moskaluk, Christopher A; Fu, Zheng

    2013-01-01

    ICK/MRK (intestinal cell kinase/MAK-related kinase), MAK (male germ cell-associated kinase), and MOK (MAPK/MAK/MRK-overlapping kinase) are closely related serine/threonine protein kinases in the protein kinome. The biological functions and regulatory mechanisms of the ICK/MAK/MOK family are still largely elusive. Despite significant similarities in their catalytic domains, they diverge markedly in the sequence and structural organization of their C-terminal non-catalytic domains, raising the question as to whether they have distinct, overlapping, or redundant biological functions. In order to gain insights into their biological activities and lay a fundamental groundwork for functional studies, we investigated the spatio-temporal distribution patterns and the expression dynamics of ICK/MAK/MOK protein kinases in the intestine. We found that ICK/MAK/MOK proteins display divergent expression patterns along the duodenum-to-colon axis and during postnatal murine development. Furthermore, they are differentially partitioned between intestinal epithelium and mesenchyme. A significant increase in the protein level of ICK, but not MAK, was induced in human primary colon cancer specimens. ICK protein level was up-regulated whereas MOK protein level was down-regulated in mouse intestinal adenomas as compared with their adjacent normal intestinal mucosa. These data suggest distinct roles for ICK/MAK/MOK protein kinases in the regulation of intestinal neoplasia. Taken together, our findings demonstrate that the expressions of ICK/MAK/MOK proteins in the intestinal tract can be differentially and dynamically regulated, implicating a significant functional diversity within this group of protein kinases.

  6. Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

    PubMed Central

    del Val, Coral; White, Stephen H.

    2014-01-01

    We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

  7. Inheritance patterns of enzymes and serum proteins of mallard-black duck hybrids

    USGS Publications Warehouse

    Morgan, R.P.; Meritt, D.W.; Block, S.B.; Cole, M.A.; Sulkin, S.T.; Lee, F.B.; Henny, C.J.

    1984-01-01

    From 1974 to 1976, a breeding program was used to produce hybrids of black ducks and mallards for the evaluation of inheritance patterns of serum proteins and serum, liver and muscle enzymes. In addition to the crosses designed to produce hybrids, a series of matings in 1975 and 1976 were designed to evaluate inheritance patterns of a hybrid with either a black duck or mallard. At the F1 level, hybrids were easily distinguished using serum proteins. However, once a hybrid was crossed back to either a mallard or black duck, only 12?23% of the progeny were distinguishable from black ducks or mallards using serum proteins and 23?39% using esterases. Muscle, serum and liver enzymes were similar between the two species.

  8. Inheritance patterns of enzymes and serum proteins of mallard-black duck hybrids

    USGS Publications Warehouse

    Morgan, R.P.; Meritt, D.W.; Block, S.B.; Cole, M.

    1984-01-01

    From 1974 to 1976, a breeding program was used to produce hybrids of black ducks and mallards for the evaluation of inheritance patterns of serum proteins and serum, liver and muscle enzymes. In addition to the crosses designed to produce hybrids, a series of matings in 1975 and 1976 were designed to evaluate inheritance patterns of a hybrid with either a black duck or mallard. At the F1 level, hybrids were easily distinguished using serum proteins. However, once a hybrid was crossed back to either a mallard or black duck, only 12-23% of the progeny were distinguishable from black ducks or mallards using serum proteins and 23-39% using esterases. Muscle, serum and liver enzymes were similar between the two species.

  9. Diversity in transcripts and translational pattern of stress proteins in marine extremophiles.

    PubMed

    Ambily Nath, I V; Loka Bharathi, P A

    2011-03-01

    Extremophiles occur in a diverse range of habitats, from the frigid waters of Antarctic to the superheated plumes of hydrothermal vents. Their in-depth study could provide important insights into the biochemical, ecological and evolutionary aspects of marine microbes. The cellular machinery of such extreme-lovers could be highly flexible to cope with such harsh environments. Extreme conditions of temperature, pressure, salinity, pH, oxidative stress, radiation, etc., above the physiological tolerance level can disrupt the natural conformation of proteins in the cell. The induction of stress proteins (heat/cold shock proteins/salt stress proteins/pressure-induced proteins) plays a vital role in the acclimatization of extremophiles. The present review focuses on the in vitro studies conducted on the transcripts and translational pattern of stress proteins in extremophiles. Though some proteins are unique, a commonality in stress resistance mechanism has been observed, for example, the universal occurrence of HSP60, 70 and the expression of metabolic and DNA repair proteins. The review highlights that among all the stressful conditions, salt/osmotic stress evokes the expression of highest number of transcripts/proteins while psychrophilic condition the least.

  10. Association Between Dietary Pattern and Serum C-Reactive Protein in Japanese Men and Women

    PubMed Central

    Nanri, Hinako; Nakamura, Kazuyo; Hara, Megumi; Higaki, Yasuki; Imaizumi, Takeshi; Taguchi, Naoto; Sakamoto, Tatsuhiko; Horita, Mikako; Shinchi, Koichi; Tanaka, Keitaro

    2011-01-01

    Background Dietary pattern may influence the risks of cardiovascular disease, atherosclerosis, type 2 diabetes, and metabolic syndrome through its effects on inflammation. We evaluated the association between dietary pattern and serum high-sensitivity C-reactive protein (hs-CRP) in a Japanese population. Methods In this cross-sectional analysis, we used baseline data from 3905 men and 5640 women (age 40–69 years) who participated in a population-based cohort study between November 2005 and December 2007. Participants with possible inflammation-related diseases, current analgesic use, high hs-CRP levels (≥3000 ng/mL) or extreme dietary energy intake were excluded. We used 46 items from a validated short food frequency questionnaire and examined major dietary patterns by factor analysis. Results We identified 5 dietary patterns: healthy (high in vegetables and fruit), Western (high in meat and fried foods), seafood (high in shellfish, squid, fish, etc.), bread (high in bread and low in rice), and dessert (high in confections and fruit). After adjustment for age, alcohol use, smoking, physical activity, and body mass index, hs-CRP levels in men were inversely associated with the healthy, bread, and dessert patterns (P-trend: 0.01, 0.06, and <0.01, respectively) and positively associated with the seafood pattern (P-trend = 0.02). In women, hs-CRP levels were inversely associated with the healthy pattern (P-trend = 0.06) and positively associated with the Western pattern (P-trend = 0.06). Conclusions The healthy dietary pattern may be associated with suppressed inflammation in Japanese men and women, independently of body mass index and other factors. The sex-specific associations of hs-CRP with other dietary patterns (eg, the seafood pattern) require further study. PMID:21325731

  11. Chemistry in Microfluidic Channels

    ERIC Educational Resources Information Center

    Chia, Matthew C.; Sweeney, Christina M.; Odom, Teri W.

    2011-01-01

    General chemistry introduces principles such as acid-base chemistry, mixing, and precipitation that are usually demonstrated in bulk solutions. In this laboratory experiment, we describe how chemical reactions can be performed in a microfluidic channel to show advanced concepts such as laminar fluid flow and controlled precipitation. Three sets of…

  12. Additive-free digital microfluidics.

    PubMed

    Freire, Sergio L S; Tanner, Brendan

    2013-07-16

    Digital microfluidics, a technique for manipulation of droplets, is becoming increasingly important for the development of miniaturized platforms for laboratory processes. Despite the enthusiasm, droplet motion is frequently hindered by the desorption of proteins or other analytes to surfaces. Current approaches to minimize this unwanted surface fouling involve the addition of extra species to the droplet or its surroundings, which might be problematic depending on the droplet content. Here, a new strategy is introduced to move droplets containing cells and other analytes on solid substrates, without extra moieties; in particular, droplets with bovine serum albumin could be moved at a concentration 2000 times higher than previously reported (without additives). This capability is achieved by using a soot-based superamphiphobic surface combined with a new device geometry, which favors droplet rolling. Contrasting with electrowetting, wetting forces are not required for droplet motion.

  13. Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures

    PubMed Central

    Tesei, Donatella; Marzban, Gorji; Zakharova, Kristina; Isola, Daniela; Selbmann, Laura; Sterflinger, Katja

    2012-01-01

    Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi – Coniosporium perforans, Exophiala jeanselmei – and of the extremophilic fungus – Friedmanniomyces endolithicus – were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum. PMID:22862921

  14. Proteomic profiling reveals a severely perturbed protein expression pattern in aged skeletal muscle.

    PubMed

    O'Connell, Kathleen; Gannon, Joan; Doran, Philip; Ohlendieck, Kay

    2007-08-01

    Extended longevity is often accompanied by frailty and increased susceptibility to a variety of crippling disorders. One of the most striking features of human aging is sarcopenia, which is defined as the age-related decline in skeletal muscle mass and strength. Although various metabolic and functional defects in aging muscle fibres have been described over the last decade, it is not known whether a pathophysiological hierarchy exists within degenerative pathways leading to muscle wasting. Hence, in order to identify novel biomarkers of age-dependent skeletal muscle degeneration, we have here applied mass spectrometry-based proteomics for studying global muscle protein expression patterns. As a model system of sarcopenia, we have employed crude extracts from senescent rat gastrocnemius muscle, as compared to young adult tissue preparations. Using the highly sensitive protein dye Deep Purple for the analysis of the 2-D separated muscle proteome and peptide mass fingerprinting for the identification of individual protein spots, a differential expression pattern was observed for contractile proteins, metabolic factors, regulatory components and heat shock elements. A drastic increase was shown for alpha B-crystallin, myosin light chain MLC-1, phosphoglycerate kinase, adenylate kinase, triosephosphate isomerase, albumin, aconitase and nucleoside-diphosphate kinase in aged fibres. In contrast, the expression of pyruvate kinase, aldolase, creatine kinase, transferrin, alpha-tropomyosin and myosin light chain MLC-3 was decreased in old skeletal muscle. Comparative 2-D immunoblotting of selected candidate proteins has confirmed the effect of aging on the skeletal muscle proteome. These findings demonstrate a severely perturbed protein expression pattern in aged skeletal muscle, which reflects the underlying molecular alterations causing a drastic decline of muscle strength in the senescent organism. In the long-term, the systematic deduction of abnormal protein expression

  15. Common and distinctive localization patterns of Crumbs polarity complex proteins in the mammalian eye

    PubMed Central

    Kim, Jin Young; Song, Ji Yun; Karnam, Santi; Park, Jun Young; Lee, Jamie JH; Kim, Seonhee; Cho, Seo-Hee

    2016-01-01

    Crumbs polarity complex proteins are essential for cellular and tissue polarity, and for adhesion of epithelial cells. In epithelial tissues deletion of any of three core proteins disrupts localization of the other proteins, indicating structural and functional interdependence among core components. Despite previous studies of function and co-localization that illustrated the properties that these proteins share, it is not known whether an individual component of the complex plays a distinct role in a unique cellular and developmental context. In order to investigate this question, we primarily used confocal imaging to determine the expression and subcellular localization of the core Crumbs polarity complex proteins during ocular development. Here we show that in developing ocular tissues core Crumbs polarity complex proteins, Crb, Pals1 and Patj, generally appear in an overlapping pattern with some exceptions. All three core complex proteins localize to the apical junction of the retinal and lens epithelia. Pals1 is also localized in the Golgi of the retinal cells and Patj localizes to the nuclei of the apically located subset of progenitor cells. These findings suggest that core Crumbs polarity complex proteins exert common and independent functions depending on cellular context. PMID:25636444

  16. Electrophoretic separation of purified myelin: a method to improve the protein pattern resolving.

    PubMed

    Ravera, Silvia; Bartolucci, Martina; Barbarito, Giulia; Calzia, Daniela; Panfoli, Isabella

    2013-01-01

    Myelin sheath is a lipid-rich membrane, consisting of 70% lipid and 30% proteins, that is involved in physiological and pathological processes. For this reason its protein composition has been often investigated, principally by two-dimensional electrophoresis; however, the consistent lipid content makes it difficult to obtain good proteins separation. To improve the resolution of myelin proteins in a denaturing monodimensional gel electrophoresis, we examined several mixtures for the denaturation of the sample, utilizing different detergents and reducing agents. The definition of the protein pattern was analyzed by both "Blue Silver" Coomassie staining and Western Blot analysis against myelin basic protein, one of the most represented myelin proteins. The best resolution is observed when the sample was incubated with a mixture containing 1.25% dithiothreitol, 4 M urea, and 1% dodecyl maltoside or 1 % 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, prior to addition of denaturing agents. In conclusion, this work describes a novel method to improve the separation of myelin proteins in a monodimensional gel electrophoresis. It may be also useful for investigating other lipid-rich samples. PMID:23464917

  17. Electrophoretic separation of purified myelin: a method to improve the protein pattern resolving.

    PubMed

    Ravera, Silvia; Bartolucci, Martina; Barbarito, Giulia; Calzia, Daniela; Panfoli, Isabella

    2013-01-01

    Myelin sheath is a lipid-rich membrane, consisting of 70% lipid and 30% proteins, that is involved in physiological and pathological processes. For this reason its protein composition has been often investigated, principally by two-dimensional electrophoresis; however, the consistent lipid content makes it difficult to obtain good proteins separation. To improve the resolution of myelin proteins in a denaturing monodimensional gel electrophoresis, we examined several mixtures for the denaturation of the sample, utilizing different detergents and reducing agents. The definition of the protein pattern was analyzed by both "Blue Silver" Coomassie staining and Western Blot analysis against myelin basic protein, one of the most represented myelin proteins. The best resolution is observed when the sample was incubated with a mixture containing 1.25% dithiothreitol, 4 M urea, and 1% dodecyl maltoside or 1 % 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, prior to addition of denaturing agents. In conclusion, this work describes a novel method to improve the separation of myelin proteins in a monodimensional gel electrophoresis. It may be also useful for investigating other lipid-rich samples.

  18. Collective oscillations and coupled modes in confined microfluidic droplet arrays

    NASA Astrophysics Data System (ADS)

    Schiller, Ulf D.; Fleury, Jean-Baptiste; Seemann, Ralf; Gompper, Gerhard

    Microfluidic droplets have a wide range of applications ranging from analytic assays in cellular biology to controlled mixing in chemical engineering. Ensembles of microfluidic droplets are interesting model systems for non-equilibrium many-body phenomena. When flowing in a microchannel, trains of droplets can form microfluidic crystals whose dynamics are governed by long-range hydrodynamic interactions and boundary effects. In this contribution, excitation mechanisms for collective waves in dense and confined microfluidic droplet arrays are investigated by experiments and computer simulations. We demonstrate that distinct modes can be excited by creating specific `defect' patterns in flowing droplet trains. While longitudinal modes exhibit a short-lived cascade of pairs of laterally displacing droplets, transversely excited modes form propagating waves that behave like microfluidic phonons. We show that the confinement induces a coupling between longitudinal and transverse modes. We also investigate the life time of the collective oscillations and discuss possible mechanisms for the onset of instabilities. Our results demonstrate that microfluidic phonons can exhibit effects beyond the linear theory, which can be studied particularly well in dense and confined systems. This work was supported by Deutsche Forschungsgemeinschaft under Grant No. SE 1118/4.

  19. Cell-based bioassays in microfluidic systems

    NASA Astrophysics Data System (ADS)

    Itle, Laura J.; Zguris, Jeanna C.; Pishko, Michael V.

    2004-12-01

    The development of cell-based bioassays for high throughput drug screening or the sensing of biotoxins is contingent on the development of whole cell sensors for specific changes in intracellular conditions and the integration of those systems into sample delivery devices. Here we show the feasibility of using a 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate, a fluorescent dye capable of responding to changes in intracellular pH, as a detection method for the bacterial endotoxin, lipopolysaccharide. We used photolithography to entrap cells with this dye within poly(ethylene) glyocol diacrylate hydrogels in microfluidic channels. After 18 hours of exposure to lipopolysaccharide, we were able to see visible changes in the fluorescent pattern. This work shows the feasibility of using whole cell based biosensors within microfluidic networks to detect cellular changes in response to exogenous agents.

  20. Proteins linked to autosomal dominant and autosomal recessive disorders harbor characteristic rare missense mutation distribution patterns.

    PubMed

    Turner, Tychele N; Douville, Christopher; Kim, Dewey; Stenson, Peter D; Cooper, David N; Chakravarti, Aravinda; Karchin, Rachel

    2015-11-01

    The role of rare missense variants in disease causation remains difficult to interpret. We explore whether the clustering pattern of rare missense variants (MAF < 0.01) in a protein is associated with mode of inheritance. Mutations in genes associated with autosomal dominant (AD) conditions are known to result in either loss or gain of function, whereas mutations in genes associated with autosomal recessive (AR) conditions invariably result in loss-of-function. Loss-of-function mutations tend to be distributed uniformly along protein sequence, whereas gain-of-function mutations tend to localize to key regions. It has not previously been ascertained whether these patterns hold in general for rare missense mutations. We consider the extent to which rare missense variants are located within annotated protein domains and whether they form clusters, using a new unbiased method called CLUstering by Mutation Position. These approaches quantified a significant difference in clustering between AD and AR diseases. Proteins linked to AD diseases exhibited more clustering of rare missense mutations than those linked to AR diseases (Wilcoxon P = 5.7 × 10(-4), permutation P = 8.4 × 10(-4)). Rare missense mutation in proteins linked to either AD or AR diseases was more clustered than controls (1000G) (Wilcoxon P = 2.8 × 10(-15) for AD and P = 4.5 × 10(-4) for AR, permutation P = 3.1 × 10(-12) for AD and P = 0.03 for AR). The differences in clustering patterns persisted even after removal of the most prominent genes. Testing for such non-random patterns may reveal novel aspects of disease etiology in large sample studies. PMID:26246501

  1. Analysis of evolutionary conservation patterns and their influence on identifying protein functional sites.

    PubMed

    Fang, Chun; Noguchi, Tamotsu; Yamana, Hayato

    2014-10-01

    Evolutionary conservation information included in position-specific scoring matrix (PSSM) has been widely adopted by sequence-based methods for identifying protein functional sites, because all functional sites, whether in ordered or disordered proteins, are found to be conserved at some extent. However, different functional sites have different conservation patterns, some of them are linear contextual, some of them are mingled with highly variable residues, and some others seem to be conserved independently. Every value in PSSMs is calculated independently of each other, without carrying the contextual information of residues in the sequence. Therefore, adopting the direct output of PSSM for prediction fails to consider the relationship between conservation patterns of residues and the distribution of conservation scores in PSSMs. In order to demonstrate the importance of combining PSSMs with the specific conservation patterns of functional sites for prediction, three different PSSM-based methods for identifying three kinds of functional sites have been analyzed. Results suggest that, different PSSM-based methods differ in their capability to identify different patterns of functional sites, and better combining PSSMs with the specific conservation patterns of residues would largely facilitate the prediction.

  2. Biomimetic Polymer Brushes Containing Tethered Acetylcholine Analogs for Protein and Hippocampal Neuronal Cell Patterning

    PubMed Central

    Zhou, Zhaoli; Yu, Panpan; Geller, Herbert M.; Ober, Christopher K.

    2013-01-01

    This paper describes a method to control neuronal cell adhesion and differentiation with both chemical and topographic cues by using a spatially defined polymer brush pattern. First, biomimetic methacrylate polymer brushes containing tethered neurotransmitter acetylcholine functionalities in the form of dimethylaminoethyl methacrylate (DMAEMA), or free hydroxyl-terminated poly(ethylene glycol) (PEG) units were prepared using the “grown from” method through surface-initiated atom transfer radical polymerization (SI-ATRP) reactions. The surface properties of the resulting brushes were thoroughly characterized with various techniques and hippocampal neuronal cell culture on the brush surfaces exhibit cell viability and differentiation comparable to, or even better than, those on commonly used poly-L-lysine coated glass coverslips. The polymer brushes were then patterned via UV photolithography techniques to provide specially designed surface features with different sizes (varying from 2 µm to 200 µm) and orientations (horizontal and vertical). Protein absorption experiments and hippocampal neuronal cell culture tests on the brush patterns showed that both protein and neurons can adhere to the patterns and therefore be guided by such patterns. These results also demonstrate that, because of their unique chemical composition and well-defined nature, the developed polymer brushes may find many potential applications in cell-material interactions studies and neural tissue engineering. PMID:23336729

  3. Direct digital manufacturing of autonomous centrifugal microfluidic device

    NASA Astrophysics Data System (ADS)

    Ukita, Yoshiaki; Takamura, Yuzuru; Utsumi, Yuichi

    2016-06-01

    This paper presents strategies that attempt to solve two key problems facing the commercialization of microfluidics: cost reduction in microfluidic chip manufacturing and microfluidic device driver development. To reduce the cost of microfluidic chip manufacturing, we propose to use of three-dimensional (3D) printers for direct digital manufacturing (DDM). An evaluation of 3D micro-scale structure printing using several 3D printers is reported, and some of the technical issues to be addressed in the future are suggested. To evaluate micro-scale printing, three types of 3D printers, with the ability to print structures on the scale of several hundred meters, were selected by first screening six 3D printers. Line and space patterns with line widths of 100–500 µm and an aspect ratio of one were printed and evaluated. The estimated critical dimension was around 200 µm. The manufacturing of a monolithic microfluidic chip with embedded channels was also demonstrated. Monolithic microfluidic chips with embedded microchannels having 500 × 500 and 250 × 250 µm2 cross sections and 2–20 mm lengths were printed, and the fidelity of the channel shape, residual supporting material, and flow of liquid water were evaluated. The liquid flow evaluation showed that liquid water could flow through all of the microchannels with the 500 × 500 µm2 cross section, whereas this was not possible through some of the channels with the 250 × 250 µm2 cross section because of the residual resin or supporting material. To reduce the device-driver cost, we propose to use of the centrifugal microfluidic concept. An autonomous microfluidic device that could implement sequential flow control under a steadily rotating condition was printed. Four-step flow injection under a steadily rotating condition at 1500 rpm was successfully demonstrated without any external triggering such as changing the rotational speed.

  4. Direct digital manufacturing of autonomous centrifugal microfluidic device

    NASA Astrophysics Data System (ADS)

    Ukita, Yoshiaki; Takamura, Yuzuru; Utsumi, Yuichi

    2016-06-01

    This paper presents strategies that attempt to solve two key problems facing the commercialization of microfluidics: cost reduction in microfluidic chip manufacturing and microfluidic device driver development. To reduce the cost of microfluidic chip manufacturing, we propose to use of three-dimensional (3D) printers for direct digital manufacturing (DDM). An evaluation of 3D micro-scale structure printing using several 3D printers is reported, and some of the technical issues to be addressed in the future are suggested. To evaluate micro-scale printing, three types of 3D printers, with the ability to print structures on the scale of several hundred meters, were selected by first screening six 3D printers. Line and space patterns with line widths of 100-500 µm and an aspect ratio of one were printed and evaluated. The estimated critical dimension was around 200 µm. The manufacturing of a monolithic microfluidic chip with embedded channels was also demonstrated. Monolithic microfluidic chips with embedded microchannels having 500 × 500 and 250 × 250 µm2 cross sections and 2-20 mm lengths were printed, and the fidelity of the channel shape, residual supporting material, and flow of liquid water were evaluated. The liquid flow evaluation showed that liquid water could flow through all of the microchannels with the 500 × 500 µm2 cross section, whereas this was not possible through some of the channels with the 250 × 250 µm2 cross section because of the residual resin or supporting material. To reduce the device-driver cost, we propose to use of the centrifugal microfluidic concept. An autonomous microfluidic device that could implement sequential flow control under a steadily rotating condition was printed. Four-step flow injection under a steadily rotating condition at 1500 rpm was successfully demonstrated without any external triggering such as changing the rotational speed.

  5. Comparative study of electrophoretic patterns of latex proteins from clones of Hevea brasiliensis.

    PubMed

    Arreguín, B; Lara, P; Rodríguez, R

    1988-07-01

    Latex serum proteins from Hevea brasiliensis were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins from whole serum and fractions isolated by gel chromatography on Ultrogel AcA 44 were analyzed. No qualitative clonal differences were found in the protein patterns of whole latex or in the fractions but laser densitometry revealed reliable quantitative differences in protein composition. Reproducible mobilities and molecular weights of selected bands were obtained both within single gels as well as in different gels, analyzing several lots of latex received at various times from a Hevea experimental field station. The clones compared were IAN 710, GV 31, GV 42; the first one had the highest rubber yields. PMID:3234370

  6. Protein interaction patterns in different cellular environments are revealed by in-cell NMR

    PubMed Central

    Barbieri, Letizia; Luchinat, Enrico; Banci, Lucia

    2015-01-01

    In-cell NMR allows obtaining atomic-level information on biological macromolecules in their physiological environment. Soluble proteins may interact with the cellular environment in different ways: either specifically, with their functional partners, or non-specifically, with other cellular components. Such behaviour often causes the disappearance of the NMR signals. Here we show that by introducing mutations on the human protein profilin 1, used here as a test case, the in-cell NMR signals can be recovered. In human cells both specific and non-specific interactions are present, while in bacterial cells only the effect of non-specific interactions is observed. By comparing the NMR signal recovery pattern in human and bacterial cells, the relative contribution of each type of interaction can be assessed. This strategy allows detecting solution in-cell NMR spectra of soluble proteins without altering their fold, thus extending the applicability of in-cell NMR to a wider range of proteins. PMID:26399546

  7. Sex dependent alterations in the protein characterization patterns of Haemonchus contortus.

    PubMed

    Jaiswal, Amit Kumar; Sudan, Vikrant; Pandey, Vijay; Singh, Amit; Gaur, Ruchi Singh; Kanojiya, Dharmendra; Nigam, Rajesh; Shanker, Daya

    2016-09-01

    The aim of the study was to highlight the sex dependent differences in the electrophoretic protein patterns of male and female Haemonchus contortus worms SDS based polyacrylamide gels of both male and female worms were run side by side for comparison. A total of 33 and 35 polypeptides were detected in polyacrylamide gels stained with coomassie brilliant blue R-250, respectively. Besides many of the fundamental homologies in protein profile, some of the polypeptides specific to either sex were also observed. Most of the characteristic polypeptides were of low molecular weight. These polypeptides needs deeper unrevealing regarding the nature of protein, through well planned zymographic studies, so as to ascertain the true nature and/or type of protein involved in those bands. This will help us in better understanding of parasite immunology and sex influenced differences amongst the worm and the possible variations in their pathogenesis contributed thereof, if any. PMID:27605828

  8. Developmental pattern of rat intestinal brush-border enzymic proteins along the villus--crypt axis.

    PubMed

    Simon, P M; Kedinger, M; Raul, F; Grenier, J F; Haffen, K

    1979-02-15

    At various postnatal stages, intestinal epithelial cells were isolated sequentially from villus tip to crypt base by successive EDTA treatments. According to the localization of marker enzymic activities, isolated cells were pooled into three cell compartments: villus (V), lower villus and upper crypt (VC) and crypt (C). Purified brush-border-membrane proteins were separated by 7.5%-polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Enzymic activities could be assigned to some protein bands: maltase/glucoamylase (protein band 3), sucrase-isomaltase (protein bands 3 and 6), lactase (protein band 5) and alkaline phosphatase (region of protein bands 8 and 9). The findings suggest the following. (1) Sucrase-isomaltase activities appeared in compartment C at 17 days with a simultaneous increase of the pre-existing protein band 3 and appearance of a well-defined protein band in position 6; the enzymic complex remained still present in the crypt cells until adulthood. From the day 21 onwards, sucrase-isomaltase was detected in compartments VC and V. (2) Lactase was only present in the three cell compartments until day 21; at this developmental stage its activity completely disappeared from compartment C, in spite of the persistence of a weak protein band. (3) Alkaline phosphatase activity could be detected as a single peak corresponding to protein band 9 in all three cell compartments until day 21; thereafter it was replaced by two peaks of activity showing a less precise correlation with the well-defined protein bands 8 and 9. In the crypt cells of the adult rat, however, the preweaning situation, which was regularly observed, is an unexpected phenomenon. (4) Maltase and glucoamylase did not display any marked qualitative or quantitative modifications either along the villus-crypt axis or during the period of postnatal development studied. Evidence is given from the present data that each brush-border enzyme investigated has a specific

  9. Simple and cheap microfluidic devices for the preparation of monodisperse emulsions.

    PubMed

    Deng, Nan-Nan; Meng, Zhi-Jun; Xie, Rui; Ju, Xiao-Jie; Mou, Chuan-Lin; Wang, Wei; Chu, Liang-Ying

    2011-12-01

    Droplet microfluidics, which can generate monodisperse droplets or bubbles in unlimited numbers, at high speed and with complex structures, have been extensively investigated in chemical and biological fields. However, most current methods for fabricating microfluidic devices, such as glass etching, soft lithography in polydimethylsiloxane (PDMS) or assembly of glass capillaries, are usually either expensive or complicated. Here we report the fabrication of simple and cheap microfluidic devices based on patterned coverslips and microscope glass slides. The advantages of our approach for fabricating microfluidic devices lie in a simple process, inexpensive processing equipment and economical laboratory supplies. The fabricated microfluidic devices feature a flexible design of microchannels, easy spatial patterning of surface wettability, and good chemical compatibility and optical properties. We demonstrate their utilities for generation of monodisperse single and double emulsions with highly controllable flexibility. PMID:22025190

  10. Droplet microfluidics based microseparation systems.

    PubMed

    Xiao, Zhiliang; Niu, Menglei; Zhang, Bo

    2012-06-01

    Lab on a chip (LOC) technology is a promising miniaturization approach. The feature that it significantly reduced sample consumption makes great sense in analytical and bioanalytical chemistry. Since the start of LOC technology, much attention has been focused on continuous flow microfluidic systems. At the turn of the century, droplet microfluidics, which was also termed segmented flow microfluidics, was introduced. Droplet microfluidics employs two immiscible phases to form discrete droplets, which are ideal vessels with confined volume, restricted dispersion, limited cross-contamination, and high surface area. Due to these unique features, droplet microfluidics proves to be a versatile tool in microscale sample handling. This article reviews the utility of droplet microfluidics in microanalytical systems with an emphasize on separation science, including sample encapsulation at ultra-small volume, compartmentalization of separation bands, isolation of droplet contents, and related detection techniques.

  11. Centrifugal microfluidic platforms: advanced unit operations and applications.

    PubMed

    Strohmeier, O; Keller, M; Schwemmer, F; Zehnle, S; Mark, D; von Stetten, F; Zengerle, R; Paust, N

    2015-10-01

    Centrifugal microfluidics has evolved into a mature technology. Several major diagnostic companies either have products on the market or are currently evaluating centrifugal microfluidics for product development. The fields of application are widespread and include clinical chemistry, immunodiagnostics and protein analysis, cell handling, molecular diagnostics, as well as food, water, and soil analysis. Nevertheless, new fluidic functions and applications that expand the possibilities of centrifugal microfluidics are being introduced at a high pace. In this review, we first present an up-to-date comprehensive overview of centrifugal microfluidic unit operations. Then, we introduce the term "process chain" to review how these unit operations can be combined for the automation of laboratory workflows. Such aggregation of basic functionalities enables efficient fluidic design at a higher level of integration. Furthermore, we analyze how novel, ground-breaking unit operations may foster the integration of more complex applications. Among these are the storage of pneumatic energy to realize complex switching sequences or to pump liquids radially inward, as well as the complete pre-storage and release of reagents. In this context, centrifugal microfluidics provides major advantages over other microfluidic actuation principles: the pulse-free inertial liquid propulsion provided by centrifugal microfluidics allows for closed fluidic systems that are free of any interfaces to external pumps. Processed volumes are easily scalable from nanoliters to milliliters. Volume forces can be adjusted by rotation and thus, even for very small volumes, surface forces may easily be overcome in the centrifugal gravity field which enables the efficient separation of nanoliter volumes from channels, chambers or sensor matrixes as well as the removal of any disturbing bubbles. In summary, centrifugal microfluidics takes advantage of a comprehensive set of fluidic unit operations such as

  12. Centrifugal microfluidic platforms: advanced unit operations and applications.

    PubMed

    Strohmeier, O; Keller, M; Schwemmer, F; Zehnle, S; Mark, D; von Stetten, F; Zengerle, R; Paust, N

    2015-10-01

    Centrifugal microfluidics has evolved into a mature technology. Several major diagnostic companies either have products on the market or are currently evaluating centrifugal microfluidics for product development. The fields of application are widespread and include clinical chemistry, immunodiagnostics and protein analysis, cell handling, molecular diagnostics, as well as food, water, and soil analysis. Nevertheless, new fluidic functions and applications that expand the possibilities of centrifugal microfluidics are being introduced at a high pace. In this review, we first present an up-to-date comprehensive overview of centrifugal microfluidic unit operations. Then, we introduce the term "process chain" to review how these unit operations can be combined for the automation of laboratory workflows. Such aggregation of basic functionalities enables efficient fluidic design at a higher level of integration. Furthermore, we analyze how novel, ground-breaking unit operations may foster the integration of more complex applications. Among these are the storage of pneumatic energy to realize complex switching sequences or to pump liquids radially inward, as well as the complete pre-storage and release of reagents. In this context, centrifugal microfluidics provides major advantages over other microfluidic actuation principles: the pulse-free inertial liquid propulsion provided by centrifugal microfluidics allows for closed fluidic systems that are free of any interfaces to external pumps. Processed volumes are easily scalable from nanoliters to milliliters. Volume forces can be adjusted by rotation and thus, even for very small volumes, surface forces may easily be overcome in the centrifugal gravity field which enables the efficient separation of nanoliter volumes from channels, chambers or sensor matrixes as well as the removal of any disturbing bubbles. In summary, centrifugal microfluidics takes advantage of a comprehensive set of fluidic unit operations such as

  13. Mapping protein abundance patterns in the brain using voxelation combined with liquid chromatography and mass spectrometry

    SciTech Connect

    Petyuk, Vladislav A.; Qian, Weijun; Smith, Richard D.; Smith, Desmond J.

    2010-02-01

    Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps

  14. Microfluidic Chips for Semen Analysis

    PubMed Central

    Segerink, L.I.; Sprenkels, A.J.; Oosterhuis, G.J.E.; Vermes, I.; van den Berg, A.

    2012-01-01

    The gold standard of semen analysis is still an manual method, which is time-consuming, labour intensive and needs thorough quality control. Microfluidics can also offer advantages for this application. Therefore a first step in the development of a microfluidic chip has been made, which enables the man the semen analysis at home. In this article recent efforts to determine the concentration and motility using a microfluidic chip are summarized.

  15. Microfluidic Electroporation for Cellular Analysis and Delivery

    PubMed Central

    Geng, Tao

    2013-01-01

    Electroporation is a simple yet powerful technique for breaching cell membrane barrier. The applications of electroporation can be generally divided into two categories: the release of intracellular proteins, nucleic acids and other metabolites for analysis and the delivery of exogenous reagents such as genes, drugs and nanoparticles with therapeutic purposes or for cellular manipulation. In this review, we go over the basic physics associated with cell electroporation and highlight recent technological advances on microfluidic platforms for conducting electroporation. Within the context of its working mechanism, we summarize the accumulated knowledge on how the parameters of electroporation affect its performance for various tasks. We discuss various strategies and designs for conducting electroporation at microscale and then focus on analysis of intracellular contents and delivery of exogenous agents as two major applications of the technique. Finally, an outlook for future applications of microfluidic electroporation in increasingly diverse utilities is presented. PMID:23917998

  16. Microfluidic electroporation for cellular analysis and delivery.

    PubMed

    Geng, Tao; Lu, Chang

    2013-10-01

    Electroporation is a simple yet powerful technique for breaching the cell membrane barrier. The applications of electroporation can be generally divided into two categories: the release of intracellular proteins, nucleic acids and other metabolites for analysis and the delivery of exogenous reagents such as genes, drugs and nanoparticles with therapeutic purposes or for cellular manipulation. In this review, we go over the basic physics associated with cell electroporation and highlight recent technological advances on microfluidic platforms for conducting electroporation. Within the context of its working mechanism, we summarize the accumulated knowledge on how the parameters of electroporation affect its performance for various tasks. We discuss various strategies and designs for conducting electroporation at the microscale and then focus on analysis of intracellular contents and delivery of exogenous agents as two major applications of the technique. Finally, an outlook for future applications of microfluidic electroporation in increasingly diverse utilities is presented.

  17. Analysis of the expression patterns, subcellular localisations and interaction partners of Drosophila proteins using a pigP protein trap library

    PubMed Central

    Lowe, Nick; Rees, Johanna S.; Roote, John; Ryder, Ed; Armean, Irina M.; Johnson, Glynnis; Drummond, Emma; Spriggs, Helen; Drummond, Jenny; Magbanua, Jose P.; Naylor, Huw; Sanson, Bénédicte; Bastock, Rebecca; Huelsmann, Sven; Trovisco, Vitor; Landgraf, Matthias; Knowles-Barley, Seymour; Armstrong, J. Douglas; White-Cooper, Helen; Hansen, Celia; Phillips, Roger G.; Lilley, Kathryn S.; Russell, Steven; St Johnston, Daniel

    2014-01-01

    Although we now have a wealth of information on the transcription patterns of all the genes in the Drosophila genome, much less is known about the properties of the encoded proteins. To provide information on the expression patterns and subcellular localisations of many proteins in parallel, we have performed a large-scale protein trap screen using a hybrid piggyBac vector carrying an artificial exon encoding yellow fluorescent protein (YFP) and protein affinity tags. From screening 41 million embryos, we recovered 616 verified independent YFP-positive lines representing protein traps in 374 genes, two-thirds of which had not been tagged in previous P element protein trap screens. Over 20 different research groups then characterized the expression patterns of the tagged proteins in a variety of tissues and at several developmental stages. In parallel, we purified many of the tagged proteins from embryos using the affinity tags and identified co-purifying proteins by mass spectrometry. The fly stocks are publicly available through the Kyoto Drosophila Genetics Resource Center. All our data are available via an open access database (Flannotator), which provides comprehensive information on the expression patterns, subcellular localisations and in vivo interaction partners of the trapped proteins. Our resource substantially increases the number of available protein traps in Drosophila and identifies new markers for cellular organelles and structures. PMID:25294943

  18. Rapid fabrication of supercapacitor electrodes using bionanoscaffolds in capillary microfluidics

    NASA Astrophysics Data System (ADS)

    Zang, F.; Chu, S.; Gerasopoulos, K.; Culver, J. N.; Ghodssi, R.

    2015-12-01

    This paper reports the utilization of capillary microfluidics to rapidly create nanostructure-patterned electrodes for energy storage applications. Using patterned photoresist as open-channel capillary microfluidics, Tobacco mosaic virus (TMV) bio-nanoscaffolds suspended in solution are autonomously delivered onto planar gold electrodes over a 1 cm2 area. The TMVs assemble on the electrode and form a dense bio-nanoscaffold layer due to enhanced evaporation-assisted assembly in the open-channel capillary microfluidic device within an hour. The TMV structures are coated with Ni/NiO through electroless plating and thermal oxidation to form supercapacitor electrodes. The galvanostatic charge/discharge cycle showed a 3.6-fold increase in areal capacitance for the nanostructured electrode compared to planar structures.

  19. Unexpected Deposition Patterns of Recombinant Proteins in Post-Endoplasmic Reticulum Compartments of Wheat Endosperm1

    PubMed Central

    Arcalis, Elsa; Marcel, Sylvain; Altmann, Friedrich; Kolarich, Daniel; Drakakaki, Georgia; Fischer, Rainer; Christou, Paul; Stoger, Eva

    2004-01-01

    Protein transport within cereal endosperm cells is complicated by the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. For wheat storage proteins, two major transport routes run from the ER to the vacuole, one bypassing and one passing through the Golgi. Proteins traveling along each route converge at the vacuole and form aggregates. To determine the impact of this trafficking system on the fate of recombinant proteins expressed in wheat endosperm, we used confocal and electron microscopy to investigate the fate of three recombinant proteins containing different targeting information. KDEL-tagged recombinant human serum albumin, which is retrieved to the ER lumen in leaf cells, was deposited in prolamin aggregates within the vacuole of endosperm cells, most likely following the bulk of endogenous glutenins. Recombinant fungal phytase, a glycoprotein designed for secretion, was delivered to the same compartment, with no trace of the molecule in the apoplast. Glycan analysis revealed that this protein had passed through the Golgi. The localization of human serum albumin and phytase was compared to that of recombinant legumin, which contains structural targeting information directing it to the vacuole. Uniquely, legumin accumulated in the globulin inclusion bodies at the periphery of the prolamin bodies, suggesting a different mode of transport and/or aggregation. Our results demonstrate that recombinant proteins are deposited in an unexpected pattern within wheat endosperm cells, probably because of the unique storage properties of this tissue. Our data also confirm that recombinant proteins are invaluable tools for the analysis of protein trafficking in cereals. PMID:15489278

  20. Effect of altered eating pattern on serum fructosamine: total protein ratio and plasma glucose level.

    PubMed

    Ch'ng, S L; Cheah, S H; Husain, R; Duncan, M T

    1989-05-01

    The effect of alteration of eating pattern during Ramadan on body mass index (BMI), serum fructosamine: total protein ratio (F/TP), and glucose level in 18 healthy male Asiatic Moslems were studied. The results showed a significant decrease (p less than 0.025) in F/TP at the second week of Ramadan in 11 subjects who experienced continuous decrease in BMI throughout Ramadan. The remaining 7 subjects showed no significant changes in BMI and F/TP. No evidence of hypoglycaemia was observed in the subjects during the study. Serum fructosamine: total protein ratio in subjects with altered eating pattern preferably should be interpreted along with the change in body mass index.

  1. Phylogenetic Analyses in Dorylaimida Using Data from 2-D Protein Patterns

    PubMed Central

    Ferris, V. R.; Ferris, J. M.

    1988-01-01

    Data from two-dimensional protein patterns for nine dorylaimid isolates were analyzed using PAUP, a computer program for inferring phylogenies under the principle of maximum parsimony. With a variety of available options, including branch swapping and rooting, essentially the same tree was obtained. When isolates of the genus Labronema were analyzed alone, all trees obtained had the same topology, although tree length varied considerably, depending on whether a hypothetical ancestral taxon was included. PMID:19290191

  2. Conversion of amino-acid sequence in proteins to classical music: search for auditory patterns

    PubMed Central

    2007-01-01

    We have converted genome-encoded protein sequences into musical notes to reveal auditory patterns without compromising musicality. We derived a reduced range of 13 base notes by pairing similar amino acids and distinguishing them using variations of three-note chords and codon distribution to dictate rhythm. The conversion will help make genomic coding sequences more approachable for the general public, young children, and vision-impaired scientists. PMID:17477882

  3. Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals

    NASA Astrophysics Data System (ADS)

    Kirian, R. A.; White, T. A.; Holton, J. M.; Chapman, H. N.; Fromme, P.; Barty, A.; Lomb, L.; Aquila, A.

    2011-03-01

    A complete set of structure factors has been extracted from hundreds of thousands of femtosecond X-ray diffraction patterns from randomly oriented Photosystem I membrane protein nanocrystals, using the Monte Carlo method of intensity integration. The data, collected at the Linac Coherent Light Source, are compared with conventional single-crystal data collected at a synchrotron source, and the quality of each data set was found to be similar.

  4. Macromolecular Crystal Growth by Means of Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark; Ferree, Darren; Spearing, Scott; Monaco, Lisa; Molho, Josh; Spaid, Michael; Brasseur, Mike; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    We have performed a feasibility study in which we show that chip-based, microfluidic (LabChip(TM)) technology is suitable for protein crystal growth. This technology allows for accurate and reliable dispensing and mixing of very small volumes while minimizing bubble formation in the crystallization mixture. The amount of (protein) solution remaining after completion of an experiment is minimal, which makes this technique efficient and attractive for use with proteins, which are difficult or expensive to obtain. The nature of LabChip(TM) technology renders it highly amenable to automation. Protein crystals obtained in our initial feasibility studies were of excellent quality as determined by X-ray diffraction. Subsequent to the feasibility study, we designed and produced the first LabChip(TM) device specifically for protein crystallization in batch mode. It can reliably dispense and mix from a range of solution constituents into two independent growth wells. We are currently testing this design to prove its efficacy for protein crystallization optimization experiments. In the near future we will expand our design to incorporate up to 10 growth wells per LabChip(TM) device. Upon completion, additional crystallization techniques such as vapor diffusion and liquid-liquid diffusion will be accommodated. Macromolecular crystallization using microfluidic technology is envisioned as a fully automated system, which will use the 'tele-science' concept of remote operation and will be developed into a research facility for the International Space Station as well as on the ground.

  5. Surface Acoustic Wave Microfluidics

    NASA Astrophysics Data System (ADS)

    Yeo, Leslie Y.; Friend, James R.

    2014-01-01

    Fluid manipulations at the microscale and beyond are powerfully enabled through the use of 10-1,000-MHz acoustic waves. A superior alternative in many cases to other microfluidic actuation techniques, such high-frequency acoustics is almost universally produced by surface acoustic wave devices that employ electromechanical transduction in wafer-scale or thin-film piezoelectric media to generate the kinetic energy needed to transport and manipulate fluids placed in adjacent microfluidic structures. These waves are responsible for a diverse range of complex fluid transport phenomena - from interfacial fluid vibration and drop and confined fluid transport to jetting and atomization - underlying a flourishing research literature spanning fundamental fluid physics to chip-scale engineering applications. We highlight some of this literature to provide the reader with a historical basis, routes for more detailed study, and an impression of the field's future directions.

  6. Microfluidic colloid filtration

    PubMed Central

    Linkhorst, John; Beckmann, Torsten; Go, Dennis; Kuehne, Alexander J. C.; Wessling, Matthias

    2016-01-01

    Filtration of natural and colloidal matter is an essential process in today’s water treatment processes. The colloidal matter is retained with the help of micro- and nanoporous synthetic membranes. Colloids are retained in a “cake layer” – often coined fouling layer. Membrane fouling is the most substantial problem in membrane filtration: colloidal and natural matter build-up leads to an increasing resistance and thus decreasing water transport rate through the membrane. Theoretical models exist to describe macroscopically the hydrodynamic resistance of such transport and rejection phenomena; however, visualization of the various phenomena occurring during colloid retention is extremely demanding. Here we present a microfluidics based methodology to follow filter cake build up as well as transport phenomena occuring inside of the fouling layer. The microfluidic colloidal filtration methodology enables the study of complex colloidal jamming, crystallization and melting processes as well as translocation at the single particle level. PMID:26927706

  7. Microfluidic redox battery.

    PubMed

    Lee, Jin Wook; Goulet, Marc-Antoni; Kjeang, Erik

    2013-07-01

    A miniaturized microfluidic battery is proposed, which is the first membraneless redox battery demonstrated to date. This unique concept capitalizes on dual-pass flow-through porous electrodes combined with stratified, co-laminar flow to generate electrical power on-chip. The fluidic design is symmetric to allow for both charging and discharging operations in forward, reverse, and recirculation modes. The proof-of-concept device fabricated using low-cost materials integrated in a microfluidic chip is shown to produce competitive power levels when operated on a vanadium redox electrolyte. A complete charge/discharge cycle is performed to demonstrate its operation as a rechargeable battery, which is an important step towards providing sustainable power to lab-on-a-chip and microelectronic applications. PMID:23712370

  8. Microfluidic channel fabrication method

    DOEpatents

    Arnold, Don W.; Schoeniger, Joseph S.; Cardinale, Gregory F.

    2001-01-01

    A new channel structure for microfluidic systems and process for fabricating this structure. In contrast to the conventional practice of fabricating fluid channels as trenches or grooves in a substrate, fluid channels are fabricated as thin walled raised structures on a substrate. Microfluidic devices produced in accordance with the invention are a hybrid assembly generally consisting of three layers: 1) a substrate that can or cannot be an electrical insulator; 2) a middle layer, that is an electrically conducting material and preferably silicon, forms the channel walls whose height defines the channel height, joined to and extending from the substrate; and 3) a top layer, joined to the top of the channels, that forms a cover for the channels. The channels can be defined by photolithographic techniques and are produced by etching away the material around the channel walls.

  9. Microfluidic colloid filtration

    NASA Astrophysics Data System (ADS)

    Linkhorst, John; Beckmann, Torsten; Go, Dennis; Kuehne, Alexander J. C.; Wessling, Matthias

    2016-03-01

    Filtration of natural and colloidal matter is an essential process in today’s water treatment processes. The colloidal matter is retained with the help of micro- and nanoporous synthetic membranes. Colloids are retained in a “cake layer” – often coined fouling layer. Membrane fouling is the most substantial problem in membrane filtration: colloidal and natural matter build-up leads to an increasing resistance and thus decreasing water transport rate through the membrane. Theoretical models exist to describe macroscopically the hydrodynamic resistance of such transport and rejection phenomena; however, visualization of the various phenomena occurring during colloid retention is extremely demanding. Here we present a microfluidics based methodology to follow filter cake build up as well as transport phenomena occuring inside of the fouling layer. The microfluidic colloidal filtration methodology enables the study of complex colloidal jamming, crystallization and melting processes as well as translocation at the single particle level.

  10. Microfluidic redox battery.

    PubMed

    Lee, Jin Wook; Goulet, Marc-Antoni; Kjeang, Erik

    2013-07-01

    A miniaturized microfluidic battery is proposed, which is the first membraneless redox battery demonstrated to date. This unique concept capitalizes on dual-pass flow-through porous electrodes combined with stratified, co-laminar flow to generate electrical power on-chip. The fluidic design is symmetric to allow for both charging and discharging operations in forward, reverse, and recirculation modes. The proof-of-concept device fabricated using low-cost materials integrated in a microfluidic chip is shown to produce competitive power levels when operated on a vanadium redox electrolyte. A complete charge/discharge cycle is performed to demonstrate its operation as a rechargeable battery, which is an important step towards providing sustainable power to lab-on-a-chip and microelectronic applications.

  11. Microfluidic colloid filtration.

    PubMed

    Linkhorst, John; Beckmann, Torsten; Go, Dennis; Kuehne, Alexander J C; Wessling, Matthias

    2016-01-01

    Filtration of natural and colloidal matter is an essential process in today's water treatment processes. The colloidal matter is retained with the help of micro- and nanoporous synthetic membranes. Colloids are retained in a "cake layer"--often coined fouling layer. Membrane fouling is the most substantial problem in membrane filtration: colloidal and natural matter build-up leads to an increasing resistance and thus decreasing water transport rate through the membrane. Theoretical models exist to describe macroscopically the hydrodynamic resistance of such transport and rejection phenomena; however, visualization of the various phenomena occurring during colloid retention is extremely demanding. Here we present a microfluidics based methodology to follow filter cake build up as well as transport phenomena occuring inside of the fouling layer. The microfluidic colloidal filtration methodology enables the study of complex colloidal jamming, crystallization and melting processes as well as translocation at the single particle level. PMID:26927706

  12. Zipper encodes a putative integral membrane protein required for normal axon patterning during Drosophila neurogenesis.

    PubMed Central

    Zhao, D B; Côté, S; Jähnig, F; Haller, J; Jäckle, H

    1988-01-01

    During the development of the central nervous system, Drosophila embryo axons become organized in a stereo-typed fasciculation pattern. We have found that the zipper (zip) gene, initially identified on the basis of a defective larval cuticle in zip mutant embryos, is possibly involved in the establishment or maintenance of the axon pattern during the late stages of neurogenesis. The zip wild-type gene is expressed in the developing nervous system. It codes for a putative integral membrane protein. Both the molecular features of zipper and its biological effect in the nervous system of mutants suggest that zipper is an essential component for cell surface interactions involved in axon patterning, and that the cuticle phenotype of zip mutants is dependent on the primary defects observed in the nervous system. Images PMID:3402433

  13. Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

    PubMed Central

    Bottasso Arias, Natalia M.; García, Marina; Bondar, Constanza; Guzman, Luciana; Redondo, Agustina; Chopita, Nestor; Córsico, Betina; Chirdo, Fernando G.

    2015-01-01

    Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa. PMID:26346822

  14. Accumulation of BSA in Packed-bed Microfluidics

    NASA Astrophysics Data System (ADS)

    Summers, Samantha; Hu, Chuntian; Hartman, Ryan

    2012-11-01

    Alzheimers and Parkinsons are two diseases that are associated with protein deposition in the brain, causing loss of either cognitive or muscle functioning. Protein deposition diseases are considered progressive diseases since the continual aggregation of protein causes the patient's symptoms to slowly worsen over time. There are currently no known means of treatment for protein deposition diseases. Our goal is to understand the potential for packed-bed microfluidics to study protein accumulation. Measurement of the resistance to flow through micro-scale packed-beds is critical to understanding the process of protein accumulation. Aggregation in bulk is fundamentally different from accumulation on surfaces. Our study attempts to distinguish between either mechanism. The results from our experiments involving protein injection through a microfluidic system will be presented and discussed. Funding received by NSF REU Grant 1062611.

  15. Development of a fast thermal response microfluidic system using liquid metal

    NASA Astrophysics Data System (ADS)

    Gao, Meng; Gui, Lin

    2016-07-01

    Room temperature liquid metal gallium alloy has been widely used in many micro-electromechanical systems applications, such as on-chip electrical microheaters, micro temperature sensors, micro pumps and so on. Injecting liquid metal into microchannels can provide a simple, rapid, low-cost but efficient way to integrate these elements in microfluidic chips with high accuracy. The liquid metal-filled microstructures can be designed in any shape and easily integrated into microfluidic chips. In this paper, an on-chip liquid metal-based thermal microfluidic system is proposed for quick temperature control at the microscale. The micro system utilizes just one microfluidic chip as a basic working platform, which has liquid metal-based on-chip heaters, temperature sensors and electroosmotic flow pumps. Under the comprehensive control of these elements, the micro system can quickly change the temperature of a target fluid in the microfluidic chip. These liquid metal-based on-chip elements are very helpful for the fabrication and miniaturization of the microfluidic chip. In this paper, deionized water is used to test the temperature control performance of the thermal microfluidic system. According to the experimental results, the micro system can efficiently control the temperature of water ranging from 28 °C to 90 °C. The thermal microfluidic system has great potential for use in many microfluidic applications, such as on-chip polymerase chain reaction, temperature gradient focusing, protein crystallization and chemical synthesis.

  16. Microfluidic Biochip Design

    NASA Technical Reports Server (NTRS)

    Panzarella, Charles

    2004-01-01

    As humans prepare for the exploration of our solar system, there is a growing need for miniaturized medical and environmental diagnostic devices for use on spacecrafts, especially during long-duration space missions where size and power requirements are critical. In recent years, the biochip (or Lab-on-a- Chip) has emerged as a technology that might be able to satisfy this need. In generic terms, a biochip is a miniaturized microfluidic device analogous to the electronic microchip that ushered in the digital age. It consists of tiny microfluidic channels, pumps and valves that transport small amounts of sample fluids to biosensors that can perform a variety of tests on those fluids in near real time. It has the obvious advantages of being small, lightweight, requiring less sample fluids and reagents and being more sensitive and efficient than larger devices currently in use. Some of the desired space-based applications would be to provide smaller, more robust devices for analyzing blood, saliva and urine and for testing water and food supplies for the presence of harmful contaminants and microorganisms. Our group has undertaken the goal of adapting as well as improving upon current biochip technology for use in long-duration microgravity environments. In addition to developing computational models of the microfluidic channels, valves and pumps that form the basis of every biochip, we are also trying to identify potential problems that could arise in reduced gravity and develop solutions to these problems. One such problem is due to the prevalence of bubbly sample fluids in microgravity. A bubble trapped in a microfluidic channel could be detrimental to the operation of a biochip. Therefore, the process of bubble formation in microgravity needs to be studied, and a model of this process has been developed and used to understand how bubbles develop and move through biochip components. It is clear that some type of bubble filter would be necessary in Space, and

  17. AC magnetohydrodynamic microfluidic switch

    SciTech Connect

    Lemoff, A V; Lee, A P

    2000-03-02

    A microfluidic switch has been demonstrated using an AC Magnetohydrodynamic (MHD) pumping mechanism in which the Lorentz force is used to pump an electrolytic solution. By integrating two AC MHD pumps into different arms of a Y-shaped fluidic circuit, flow can be switched between the two arms. This type of switch can be used to produce complex fluidic routing, which may have multiple applications in {micro}TAS.

  18. Laser micromachined hybrid open/paper microfluidic chips.

    PubMed

    Chumo, B; Muluneh, M; Issadore, D

    2013-01-01

    Paper-based microfluidics are an increasingly popular alternative to devices with conventional open channel geometries. The low cost of fabrication and the absence of external instrumentation needed to drive paper microchannels make them especially well suited for medical diagnostics in resource-limited settings. Despite the advantages of paper microfluidics, many assays performed using conventional open channel microfluidics are challenging to translate onto paper, such as bead, emulsion, and cell-based assays. To overcome this challenge, we have developed a hybrid open-channel/paper channel microfluidic device. In this design, wick-driven paper channels control the flow rates within conventional microfluidics. We fabricate these hybrid chips using laser-micromachined polymer sheets and filter paper. In contrast to previous efforts that utilized external, macroscopic paper-based pumps, we integrated micro-scale paper and open channels onto a single chip to control multiple open channels and control complex laminar flow-pattern within individual channels. We demonstrated that flow patterns within the open channels can be quantitatively controlled by modulating the geometry of the paper channels, and that these flow rates agree with Darcy's law. The utility of these hybrid chips, for applications such as bead-, cell-, or emulsion-based assays, was demonstrated by constructing a hybrid chip that hydrodynamically focused micrometer-sized polystyrene beads stably for >10 min, as well as cells, without external instrumentation to drive fluid flow.

  19. High-pressure microfluidics

    NASA Astrophysics Data System (ADS)

    Hjort, K.

    2015-03-01

    When using appropriate materials and microfabrication techniques, with the small dimensions the mechanical stability of microstructured devices allows for processes at high pressures without loss in safety. The largest area of applications has been demonstrated in green chemistry and bioprocesses, where extraction, synthesis and analyses often excel at high densities and high temperatures. This is accessible through high pressures. Capillary chemistry has been used since long but, just like in low-pressure applications, there are several potential advantages in using microfluidic platforms, e.g., planar isothermal set-ups, large local variations in geometries, dense form factors, small dead volumes and precisely positioned microstructures for control of reactions, catalysis, mixing and separation. Other potential applications are in, e.g., microhydraulics, exploration, gas driven vehicles, and high-pressure science. From a review of the state-of-art and frontiers of high pressure microfluidics, the focus will be on different solutions demonstrated for microfluidic handling at high pressures and challenges that remain.

  20. Multilayer microfluidic PEGDA hydrogels.

    PubMed

    Cuchiara, Michael P; Allen, Alicia C B; Chen, Theodore M; Miller, Jordan S; West, Jennifer L

    2010-07-01

    Development of robust 3D tissue analogs in vitro is limited by passive, diffusional mass transport. Perfused microfluidic tissue engineering scaffolds hold the promise to improve mass transport limitations and promote the development of complex, metabolically dense, and clinically relevant tissues. We report a simple and robust multilayer replica molding technique in which poly(dimethylsiloxane) (PDMS) and poly(ethylene glycol) diacrylate (PEGDA) are serially replica molded to develop microfluidic PEGDA hydrogel networks embedded within independently fabricated PDMS housings. We demonstrate the ability to control solute-scaffold effective diffusivity as a function of solute molecular weight and hydrogel concentration. Within cell laden microfluidic hydrogels, we demonstrate increased cellular viability in perfused hydrogel systems compared to static controls. We observed a significant increase in cell viability at all time points greater than zero at distances up to 1 mm from the perfused channel. Knowledge of spatiotemporal mass transport and cell viability gradients provides useful engineering design parameters necessary to maximize overall scaffold viability and metabolic density. This work has applications in the development of hydrogels as in vitro diagnostics and ultimately as regenerative medicine based therapeutics.

  1. Protein addressing on patterned microchip by coupling chitosan electrodeposition and 'electro-click' chemistry.

    PubMed

    Shi, Xiao-Wen; Qiu, Ling; Nie, Zhen; Xiao, Ling; Payne, Gregory F; Du, Yumin

    2013-12-01

    Many applications in proteomics and lab-on-chip analysis require methods that guide proteins to assemble at surfaces with high spatial and temporal control. Electrical inputs are particularly convenient to control, and there has been considerable effort to discover simple and generic mechanisms that allow electrical inputs to trigger protein assembly on-demand. Here, we report the electroaddressing of a protein to a patterned surface by coupling two generic electroaddressing mechanisms. First, we electrodeposit the stimuli-responsive film-forming aminopolysaccharide chitosan to form a hydrogel matrix at the electrode surface. After deposition, the matrix is chemically functionalized with alkyne groups. Second, we ''electro-click' an azide-tagged protein to the functionalized matrix using electrical signals to trigger conjugation by Huisgen 1,3-dipolar cycloadditions. Specifically, a cathodic potential is applied to the matrix-coated electrode to reduce Cu(II) to Cu(I) which is required for the click reaction. Using fluorescently-labeled bovine serum albumin as our model, we demonstrate that protein conjugation can be controlled spatially and temporally. We anticipate that the coupling of polysaccharide electrodeposition and electro-click chemistry will provide a simple and generic approach to electroaddress proteins within compatible hydrogel matrices.

  2. Protein and alkaloid patterns of the floral nectar in some solanaceous species.

    PubMed

    Kerchner, András; Darók, Judit; Bacskay, Ivett; Felinger, Attila; Jakab, Gábor; Farkas, Ágnes

    2015-09-01

    The family Solanaceae includes several melliferous plants, which tend to produce copious amounts of nectar. Floral nectar is a chemically complex aqueous solution, dominated by sugars, but minor components such as amino acids, proteins, flavonoids and alkaloids are present as well. This study aimed at analysing the protein and alkaloid profile of the nectar in seven solanaceous species. Proteins were examined with SDS-PAGE and alkaloids were analyzed with HPLC. The investigation of protein profile revealed significant differences in nectar-protein patterns not only between different plant genera, but also between the three Nicotiana species investigated. SDS-PAGE suggested the presence of several Nectarin proteins with antimicrobial activity in Nicotiana species. The nectar of all tobacco species contained the alkaloid nicotine, N. tabacum having the highest nicotine content. The nectar of Brugmansia suaveolens, Datura stramonium, Hyoscyamus niger and Lycium barbarum contained scopolamine, the highest content of which was measured in B. suaveolens. The alkaloid concentrations in the nectars of most solanaceous species investigated can cause deterrence in honeybees, and the nectar of N. rustica and N. tabacum can be considered toxic for honeybees. PMID:26344026

  3. Multiplexed proteomic sample preconcentration device using surface-patterned ion-selective membrane.

    PubMed

    Lee, Jeong Hoon; Song, Yong-Ak; Han, Jongyoon

    2008-04-01

    In this paper, we report a new method of fabricating a high-throughput protein preconcentrator in poly(dimethylsiloxane) (PDMS) microfluidic chip format. We print a submicron thick ion-selective membrane on the glass substrate by using standard patterning techniques. By simply plasma-bonding a PDMS microfluidic device on top of the printed glass substrate, we can integrate the ion-selective membrane into the device and rapidly prototype a PDMS preconcentrator without complicated microfabrication and cumbersome integration processes. The PDMS preconcentrator shows a concentration factor as high as approximately 10(4) in 5 min. This printing method even allows fabricating a parallel array of preconcentrators to increase the concentrated sample volume, which can facilitate an integration of our microfluidic preconcentrator chip as a signal enhancing tool to various detectors such as a mass spectrometer.

  4. Protein patterning by UV-induced photodegradation of poly(oligo(ethylene glycol) methacrylate) brushes.

    PubMed

    Alang Ahmad, Shahrul; Hucknall, Angus; Chilkoti, Ashutosh; Leggett, Graham J

    2010-06-15

    The UV photodegradation of protein-resistant poly(oligo(ethylene glycol) methacrylate) (POEGMA) bottle-brush films, grown on silicon oxide by surface-initiated atom radical transfer polymerization, was studied using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Exposure to light with a wavelength of 244 nm caused a loss of polyether units from the brush structure and the creation of aldehyde groups that could be derivatized with amines. An increase was measured in the coefficient of friction of the photodegraded polymer brush compared to the native brush, attributed to the creation of a heterogeneous surface film, leading to increased energy dissipation through film deformation and the creation of new polar functional groups at the surface. Exposure of the films through a photomask yielded sharp, well-defined patterns. Analysis of topographical images showed that physical removal of material occurred during exposure, at a rate of 1.35 nm J(-1) cm(2). Using fluorescence microscopy, the adsorption of labeled proteins onto the exposed surfaces was studied. It was found that protein strongly adsorbed to exposed areas, while the masked regions retained their protein resistance. Exposure of the film to UV light from a scanning near-field optical microscope yielded submicrometer-scale patterns. These data indicate that a simple, rapid, one-step photoconversion of the poly(OEGMA) brush occurs that transforms it from a highly protein-resistant material to one that adsorbs protein and can covalently bind amine-containing molecules and that this photoconversion can be spatially addressed with high spatial resolution.

  5. Evolutionarily Conserved Pattern of Interactions in a Protein Revealed by Local Thermal Expansion Properties.

    PubMed

    Dellarole, Mariano; Caro, Jose A; Roche, Julien; Fossat, Martin; Barthe, Philippe; García-Moreno E, Bertrand; Royer, Catherine A; Roumestand, Christian

    2015-07-29

    The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.

  6. Mixing in microfluidic devices and enhancement methods

    PubMed Central

    Ward, Kevin; Fan, Z Hugh

    2015-01-01

    Mixing in microfluidic devices presents a challenge due to laminar flows in microchannels, which result from low Reynolds numbers determined by the channel’s hydraulic diameter, flow velocity, and solution’s kinetic viscosity. To address this challenge, novel methods of mixing enhancement within microfluidic devices have been explored for a variety of applications. Passive mixing methods have been created, including those using ridges or slanted wells within the microchannels, as well as their variations with improved performance by varying geometry and patterns, by changing the properties of channel surfaces, and by optimization via simulations. In addition, active mixing methods including microstirrers, acoustic mixers, and flow pulsation have been investigated and integrated into microfluidic devices to enhance mixing in a more controllable manner. In general, passive mixers are easy to integrate, but difficult to control externally by users after fabrication. Active mixers usually take efforts to integrate within a device and they require external components (e.g. power sources) to operate. However, they can be controlled by users to a certain degree for tuned mixing. In this article, we provide a general overview of a number of passive and active mixers, discuss their advantages and disadvantages, and make suggestions on choosing a mixing method for a specific need as well as advocate possible integration of key elements of passive and active mixers to harness the advantages of both types. PMID:26549938

  7. Characterization of the pattern of ribosomal protein L19 production during the lifecycle of Leishmania spp.

    PubMed

    de Almeida-Bizzo, Janayna Hammes; Alves, Lysangela Ronalte; Castro, Felipe F; Garcia, Juliana Bório Ferreira; Goldenberg, Samuel; Cruz, Angela Kaysel

    2014-12-01

    Leishmania is a genus of protozoan parasites causing a wide clinical spectrum of diseases in humans. Analysis of a region of chromosome 6 from Leishmania major (Iribar et al.) showed that the transcript of a putative L19 ribosomal protein (RPL19) was most abundant at the amastigote stage. We therefore decided to characterize L19 protein abundance throughout the lifecycle of Leishmania. Differential expression of the L19 gene during development has been observed for all Leishmania species studied to date (L. major, L. braziliensis, L. donovani, and L. amazonensis). Immunoblotting with polyclonal antibodies against L. major RPL19 revealed that changes to L19 protein abundance follow a similar pattern in various species. The amount of L19 protein was higher in exponentially growing promastigotes than in stationary phase promastigotes. The L19 protein was barely detectable in amastigotes, despite the abundance of L19 transcripts observed in L. major at this stage. Immunofluorescence assays showed a granular, dispersed distribution of RPL19 throughout the cytoplasm. Subcellular fractionation confirmed the presence of the protein in the ribosomal fraction, but not in the cytosol of L. major. We generated a L. major transfectant bearing a plasmid-borne L19 gene. Overproduction of the L19 transcript and protein resulted in impaired growth of the transfectants in association with high polysome peaks. We also showed by metabolic labeling that L19 overexpressing clones display low rates of translation. These data suggest that L19 overexpression affects negatively translation elongation or termination. The lack of correlation between L19 transcript and protein abundances suggest that the translation of L19 is differentially controlled during development in the various species investigated.

  8. Characterization of the pattern of ribosomal protein L19 production during the lifecycle of Leishmania spp.

    PubMed

    de Almeida-Bizzo, Janayna Hammes; Alves, Lysangela Ronalte; Castro, Felipe F; Garcia, Juliana Bório Ferreira; Goldenberg, Samuel; Cruz, Angela Kaysel

    2014-12-01

    Leishmania is a genus of protozoan parasites causing a wide clinical spectrum of diseases in humans. Analysis of a region of chromosome 6 from Leishmania major (Iribar et al.) showed that the transcript of a putative L19 ribosomal protein (RPL19) was most abundant at the amastigote stage. We therefore decided to characterize L19 protein abundance throughout the lifecycle of Leishmania. Differential expression of the L19 gene during development has been observed for all Leishmania species studied to date (L. major, L. braziliensis, L. donovani, and L. amazonensis). Immunoblotting with polyclonal antibodies against L. major RPL19 revealed that changes to L19 protein abundance follow a similar pattern in various species. The amount of L19 protein was higher in exponentially growing promastigotes than in stationary phase promastigotes. The L19 protein was barely detectable in amastigotes, despite the abundance of L19 transcripts observed in L. major at this stage. Immunofluorescence assays showed a granular, dispersed distribution of RPL19 throughout the cytoplasm. Subcellular fractionation confirmed the presence of the protein in the ribosomal fraction, but not in the cytosol of L. major. We generated a L. major transfectant bearing a plasmid-borne L19 gene. Overproduction of the L19 transcript and protein resulted in impaired growth of the transfectants in association with high polysome peaks. We also showed by metabolic labeling that L19 overexpressing clones display low rates of translation. These data suggest that L19 overexpression affects negatively translation elongation or termination. The lack of correlation between L19 transcript and protein abundances suggest that the translation of L19 is differentially controlled during development in the various species investigated. PMID:25290356

  9. Site-selective adsorption of protein induced by a metal pattern on a poly(ethylene terephthalate) surface.

    PubMed

    Cao, Jiali; Wu, Zhongkui; Li, Shaoying; Tang, Hongxiao; Mei, Qilin

    2013-11-01

    A novel technique for inducing site-selective adsorption of protein through constructing metal patterns on flexible poly(ethylene terephthalate) surfaces is presented. The substrates were first modified by vacuum ultraviolet (VUV) irradiation through a photomask to introduce regions with different functional groups. Then the designed metal patterns were constructed on the surfaces of VUV-treated substrates. The surface rearrangement was effectively prevented by constructing silver patterns on poly(ethylene terephthalate) surfaces, thus significantly improving the stability and selectivity of protein adsorption on the surfaces. Moreover, the protein-repulsive layer further reinforced the effect. Finally, protein patterns were successfully obtained. As confirmed by fluorescence microscope, field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and static water contact angle measurement, the protein patterns possess both excellent selectivity and high fidelity. Feature size of the protein patterns surrounded by a protein-repulsive layer was exactly the same as that of the photomask. And the grain sizes of silver particles were approximately 50 nm. This work could potentially be used in various fields such as biomedicine, bioelectronic components, and tissue repair and replacement, where selective adsorption of protein is desired.

  10. Mapping functional group free energy patterns at protein occluded sites: nuclear receptors and G-protein coupled receptors.

    PubMed

    Lakkaraju, Sirish Kaushik; Yu, Wenbo; Raman, E Prabhu; Hershfeld, Alena V; Fang, Lei; Deshpande, Deepak A; MacKerell, Alexander D

    2015-03-23

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents.

  11. Monolithic microfluidic concentrators and mixers

    DOEpatents

    Frechet, Jean M.; Svec, Frantisek; Yu, Cong; Rohr, Thomas

    2005-05-03

    Microfluidic devices comprising porous monolithic polymer for concentration, extraction or mixing of fluids. A method for in situ preparation of monolithic polymers by in situ initiated polymerization of polymer precursors within microchannels of a microfluidic device and their use for solid phase extraction (SPE), preconcentration, concentration and mixing.

  12. Microfluidic Stamping on Sheath Flow.

    PubMed

    Yoon, Dong Hyun; Tanaka, Daiki; Sekiguchi, Tetsushi; Shoji, Shuichi

    2016-06-01

    A microfluidic stamping method to form functional shapes on a cross section in fiber-shaped flow is proposed. Microfluidic stamping and overstamping allow various cross sectional shapes on the 3D flow. The shapes can be controlled by a change in combination of structures and fluidic conditions which correspond to stamp type and stamping force. PMID:27149895

  13. Centrifugal microfluidics for biomedical applications.

    PubMed

    Gorkin, Robert; Park, Jiwoon; Siegrist, Jonathan; Amasia, Mary; Lee, Beom Seok; Park, Jong-Myeon; Kim, Jintae; Kim, Hanshin; Madou, Marc; Cho, Yoon-Kyoung

    2010-07-21

    The centrifugal microfluidic platform has been a focus of academic and industrial research efforts for almost 40 years. Primarily targeting biomedical applications, a range of assays have been adapted on the system; however, the platform has found limited commercial success as a research or clinical tool. Nonetheless, new developments in centrifugal microfluidic technologies have the potential to establish wide-spread utilization of the platform. This paper presents an in-depth review of the centrifugal microfluidic platform, while highlighting recent progress in the field and outlining the potential for future applications. An overview of centrifugal microfluidic technologies is presented, including descriptions of advantages of the platform as a microfluidic handling system and the principles behind centrifugal fluidic manipulation. The paper also discusses a history of significant centrifugal microfluidic platform developments with an explanation of the evolution of the platform as it pertains to academia and industry. Lastly, we review the few centrifugal microfluidic-based sample-to-answer analysis systems shown to date and examine the challenges to be tackled before the centrifugal platform can be more broadly accepted as a new diagnostic platform. In particular, fully integrated, easy to operate, inexpensive and accurate microfluidic tools in the area of in vitro nucleic acid diagnostics are discussed.

  14. Microfluidic paper-based biomolecule preconcentrator based on ion concentration polarization.

    PubMed

    Han, Sung Il; Hwang, Kyo Seon; Kwak, Rhokyun; Lee, Jeong Hoon

    2016-06-21

    Microfluidic paper-based analytical devices (μPADs) for molecular detection have great potential in the field of point-of-care diagnostics. Currently, a critical problem being faced by μPADs is improving their detection sensitivity. Various preconcentration processes have been developed, but they still have complicated structures and fabrication processes to integrate into μPADs. To address this issue, we have developed a novel paper-based preconcentrator utilizing ion concentration polarization (ICP) with minimal addition on lateral-flow paper. The cation selective membrane (i.e., Nafion) is patterned on adhesive tape, and this tape is then attached to paper-based channels. When an electric field is applied across the Nafion, ICP is initiated to preconcentrate the biomolecules in the paper channel. Departing from previous paper-based preconcentrators, we maintain steady lateral fluid flow with the separated Nafion layer; as a result, fluorescent dyes and proteins (FITC-albumin and bovine serum albumin) are continuously delivered to the preconcentration zone, achieving high preconcentration performance up to 1000-fold. In addition, we demonstrate that the Nafion-patterned tape can be integrated with various geometries (multiplexed preconcentrator) and platforms (string and polymer microfluidic channel). This work would facilitate integration of various ICP devices, including preconcentrators, pH/concentration modulators, and micro mixers, with steady lateral flows in paper-based platforms. PMID:27199301

  15. Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel.

    PubMed

    Choi, Jong Seob; Piao, Yunxian; Seo, Tae Seok

    2014-01-01

    The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function.

  16. Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel.

    PubMed

    Choi, Jong Seob; Piao, Yunxian; Seo, Tae Seok

    2014-01-01

    The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function. PMID:24120039

  17. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    PubMed Central

    Wu, Fabai; van Schie, Bas G.C.; Keymer, Juan E.; Dekker, Cees

    2016-01-01

    The boundary of a cell defines the shape and scale for its subcellular organisation. However, the effects of the cell’s spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shape Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2x1x1 to 11x6x1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe, and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale in adaptation to the cell size within a characteristic length range of 3–6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling, and an adaptive range, when and only when facilitated by the three-dimensional confinement of cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood. PMID:26098227

  18. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    NASA Astrophysics Data System (ADS)

    Wu, Fabai; van Schie, Bas G. C.; Keymer, Juan E.; Dekker, Cees

    2015-08-01

    The boundary of a cell defines the shape and scale of its subcellular organization. However, the effects of the cell's spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shaped Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2 × 1 × 1 to 11 × 6 × 1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale with the cell size within a characteristic length range of 3-6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling and an adaptive range, when and only when facilitated by the three-dimensional confinement of the cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood.

  19. Reconfigurable microfluidic pump enabled by opto-electrical-thermal transduction

    NASA Astrophysics Data System (ADS)

    Takeuchi, Masaru; Hagiwara, Masaya; Haulot, Gauvain; Ho, Chih-Ming

    2013-10-01

    Flexible integration of a microfluidic system comprising pumps, valves, and microchannels was realized by an optoelectronic reconfigurable microchannels (OERM) technique. Projecting a low light fluidic device pattern—e.g., pumps, valves, and channels—onto an OERM platform generates Joule heating and melts the substrate in the bright area on the platform; thus, the fluidic system can be reconfigured by changing the projected light pattern. Hexadecane was used as the substrate of the microfluidic system. The volume change of hexadecane during the liquid-solid phase transition was utilized to generate pumping pressure. The system can pump nanoliters of water within several seconds.

  20. Microfluidic vascular channels in gels using commercial 3D printers

    NASA Astrophysics Data System (ADS)

    Selvaganapathy, P. Ravi; Attalla, Rana

    2016-03-01

    This paper details the development of a three dimensional (3D) printing system with a modified microfluidic printhead used for the generation of complex vascular tissue scaffolds. The print-head features an integrated coaxial nozzle that allows the fabrication of hollow, calcium-polymerized alginate tubes that can easily be patterned using 3Dbioprinting techniques. This microfluidic design allows the incorporation of a wide range of scaffold materials as well as biological constituents such as cells, growth factors, and ECM material. With this setup, gel constructs with embedded arrays of hollow channels can be created and used as a potential substitute for blood vessel networks.

  1. An LRR-only protein representing a new type of pattern recognition receptor in Chlamys farreri.

    PubMed

    Wang, Mengqiang; Wang, Lingling; Guo, Ying; Yi, Qilin; Song, Linsheng

    2016-01-01

    Accumulating evidence has demonstrated that leucine-rich repeat (LRR)-only proteins could mediate protein-ligand and protein-protein interactions and were involved in the immune response. In the present study, an LRR-only protein (designed as CfLRRop-1) was cloned from Zhikong scallop Chlamys farreri. The complete cDNA sequence of CfLRRop-1 contained an open reading frame (ORF) of 1377 bp, which encoded a protein of 458 amino acids. An LRRNT motif, an LRR_7 motif and seven LRR motifs were found in the deduced amino acid sequence of CfLRRop-1. And these seven LRR motifs contained a conserved signature sequence LxxLxLxxNxL. The mRNA transcripts of CfLRRop-1 were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas and gonad, with the highest expression level in hepatopancreas. After the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C), the mRNA transcripts of CfLRRop-1 in haemocytes all increased firstly within the first 6 h and secondly during 12-24 h post stimulation. The mRNA expression level of CfLRRop-1 was continuously up-regulated, after the expression of CfTLR (previously identified Toll-like receptor in C. farreri) was suppressed via RNA interference (RNAi). The recombinant CfLRRop-1 protein could directly bind LPS, PGN, GLU and poly I:C, and induce the release of TNF-α in mixed primary cultured scallop haemocytes. These results collectively indicated that CfLRRop-1 would function as a powerful pattern recognition receptor (PRR) and play a pivotal role in the immune response of scallops.

  2. Electrowetting on plasma-deposited fluorocarbon hydrophobic films for biofluid transport in microfluidics

    SciTech Connect

    Bayiati, P.; Tserepi, A.; Petrou, P. S.; Kakabakos, S. E.; Misiakos, K.; Gogolides, E.

    2007-05-15

    The present work focuses on the plasma deposition of fluorocarbon (FC) films on surfaces and the electrostatic control of their wettability (electrowetting). Such films can be employed for actuation of fluid transport in microfluidic devices, when deposited over patterned electrodes. Here, the deposition was performed using C{sub 4}F{sub 8} and the plasma parameters that permit the creation of films with optimized properties desirable for electrowetting were established. The wettability of the plasma-deposited surfaces was characterized by means of contact angle measurements (in the static and dynamic mode). The thickness of the deposited films was probed in situ by means of spectroscopic ellipsometry, while the surface roughness was provided by atomic force microscopy. These plasma-deposited FC films in combination with silicon nitride, a material of high dielectric constant, were used to create a dielectric structure that requires reduced voltages for successful electrowetting. Electrowetting experiments using protein solutions were conducted on such optimized dielectric structures and were compared with similar structures bearing commercial spin-coated Teflon registered amorphous fluoropolymer (AF) film as the hydrophobic top layer. Our results show that plasma-deposited FC films have desirable electrowetting behavior and minimal protein adsorption, a requirement for successful transport of biological solutions in 'digital' microfluidics.

  3. Overexpression of an F-box protein gene disrupts cotyledon vein patterning in Arabidopsis.

    PubMed

    Cui, Xianghuan; Xu, Xiaofeng; He, Yangyang; Du, Xiling; Zhu, Jian

    2016-05-01

    Plant vascular patterning is complex. However, the detailed molecular mechanism of vascular patterning is still unknown. In this study, FBXL, an Arabidopsis F-box motif gene, was isolated by using 3' rapid amplification of cDNA ends (RACE) technique. The gene contained a coding sequence of 1407 nucleotides coding 468 amino acid residues. Amino acid sequence analysis revealed that the gene encoded a protein harboring an F-box motif at the N terminus, an LRRs motif in the middle, and an FBD motif at the C terminus. FBXL promoter-β-glucuronidase (GUS) and 35S promoter-FBXL vectors were constructed and transformed into Arabidopsis thaliana to understand the function of the FBXL gene. GUS expression analysis indicated that FBXL was specifically expressed in the vascular tissues of the root, stem, leaf, and inflorescence. FBXL overexpression in Arabidopsis displayed an abnormal venation pattern in cotyledons. Furthermore, FBXL expression was not induced by exogenous auxin and its transcript accumulation did not overlap with the distribution of endogenous auxin. These results suggested that FBXL may be involved in cotyledon vein patterning via auxin-independent pathway. PMID:26901782

  4. Coordinate control of terminal dendrite patterning and dynamics by the membrane protein Raw

    PubMed Central

    Lee, Jiae; Peng, Yun; Lin, Wen-Yang; Parrish, Jay Z.

    2015-01-01

    The directional flow of information in neurons depends on compartmentalization: dendrites receive inputs whereas axons transmit them. Axons and dendrites likewise contain structurally and functionally distinct subcompartments. Axon/dendrite compartmentalization can be attributed to neuronal polarization, but the developmental origin of subcompartments in axons and dendrites is less well understood. To identify the developmental bases for compartment-specific patterning in dendrites, we screened for mutations that affect discrete dendritic domains in Drosophila sensory neurons. From this screen, we identified mutations that affected distinct aspects of terminal dendrite development with little or no effect on major dendrite patterning. Mutation of one gene, raw, affected multiple aspects of terminal dendrite patterning, suggesting that Raw might coordinate multiple signaling pathways to shape terminal dendrite growth. Consistent with this notion, Raw localizes to branch-points and promotes dendrite stabilization together with the Tricornered (Trc) kinase via effects on cell adhesion. Raw independently influences terminal dendrite elongation through a mechanism that involves modulation of the cytoskeleton, and this pathway is likely to involve the RNA-binding protein Argonaute 1 (AGO1), as raw and AGO1 genetically interact to promote terminal dendrite growth but not adhesion. Thus, Raw defines a potential point of convergence in distinct pathways shaping terminal dendrite patterning. PMID:25480915

  5. Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography.

    PubMed

    Lau, Uland Y; Saxer, Sina S; Lee, Juneyoung; Bat, Erhan; Maynard, Heather D

    2016-01-26

    Simultaneous detection of multiple biomarkers, such as extracellular signaling molecules, is a critical aspect in disease profiling and diagnostics. Precise positioning of antibodies on surfaces, especially at the micro- and nanoscale, is important for the improvement of assays, biosensors, and diagnostics on the molecular level, and therefore, the pursuit of device miniaturization for parallel, fast, low-volume assays is a continuing challenge. Here, we describe a multiplexed cytokine immunoassay utilizing electron beam lithography and a trehalose glycopolymer as a resist for the direct writing of antibodies on silicon substrates, allowing for micro- and nanoscale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and antitumor necrosis factor alpha (TNFα) antibodies were directly patterned. Retention of the specific binding properties of the patterned antibodies was shown by the capture of secreted cytokines from stimulated RAW 264.7 macrophages. A sandwich immunoassay was employed using gold nanoparticles and enhancement with silver for the detection and visualization of bound cytokines to the patterns by localized surface plasmon resonance detected with dark-field microscopy. Multiplexing with both IL-6 and TNFα on a single chip was also successfully demonstrated with high specificity and in relevant cell culture conditions and at different times after cell stimulation. The direct fabrication of capture antibody patterns for cytokine detection described here could be useful for biosensing applications.

  6. A light writable microfluidic "flash memory": optically addressed actuator array with latched operation for microfluidic applications.

    PubMed

    Hua, Zhishan; Pal, Rohit; Srivannavit, Onnop; Burns, Mark A; Gulari, Erdogan

    2008-03-01

    This paper presents a novel optically addressed microactuator array (microfluidic "flash memory") with latched operation. Analogous to the address-data bus mediated memory address protocol in electronics, the microactuator array consists of individual phase-change based actuators addressed by localized heating through focused light patterns (address bus), which can be provided by a modified projector or high power laser pointer. A common pressure manifold (data bus) for the entire array is used to generate large deflections of the phase change actuators in the molten phase. The use of phase change material as the working media enables latched operation of the actuator array. After the initial light "writing" during which the phase is temporarily changed to molten, the actuated status is self-maintained by the solid phase of the actuator without power and pressure inputs. The microfluidic flash memory can be re-configured by a new light illumination pattern and common pressure signal. The proposed approach can achieve actuation of arbitrary units in a large-scale array without the need for complex external equipment such as solenoid valves and electrical modules, which leads to significantly simplified system implementation and compact system size. The proposed work therefore provides a flexible, energy-efficient, and low cost multiplexing solution for microfluidic applications based on physical displacements. As an example, the use of the latched microactuator array as "normally closed" or "normally open" microvalves is demonstrated. The phase-change wax is fully encapsulated and thus immune from contamination issues in fluidic environments.

  7. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography Using Surface Acoustic Waves.

    PubMed

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela H; French, Jarrod B; Huang, Tony Jun

    2015-06-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming, a surface acoustic wave-based method for manipulating and patterning crystals is developed. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and submicrometer-sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but will also make it possible to collect data on samples that were previously intractable.

  8. Nucleolar protein 4-like has a complex expression pattern in zebrafish embryos.

    PubMed

    Borah, Supriya; Barrodia, Praveen; Swain, Rajeeb K

    2016-01-01

    The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut. PMID:26934290

  9. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography using Surface Acoustic Waves

    PubMed Central

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela; French, Jarrod B.; Jun Huang, Tony

    2015-01-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming we have developed a surface acoustic wave-based method for manipulating and patterning crystals. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and sub-micrometer sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but also will make it possible to collect data on samples that were previously intractable. PMID:25641793

  10. Cancer-associated fibroblasts promote non-small cell lung cancer cell invasion by upregulation of glucose-regulated protein 78 (GRP78) expression in an integrated bionic microfluidic device.

    PubMed

    Yu, Ting; Guo, Zhe; Fan, Hui; Song, Jing; Liu, Yuanbin; Gao, Zhancheng; Wang, Qi

    2016-05-01

    The tumor microenvironment is comprised of cancer cells and various stromal cells and their respective cellular components. Cancer-associated fibroblasts (CAFs), a major part of the stromal cells, are a key determinant in tumor progression, while glucose-regulated protein (GRP)78 is overexpressed in many human cancers and is involved in tumor invasion and metastasis. This study developed a microfluidic-based three dimension (3D) co-culture device to mimic an in vitro tumor microenvironment in order to investigate tumor cell invasion in real-time. This bionic chip provided significant information regarding the role of GRP78, which may be stimulated by CAFs, to promote non-small cell lung cancer cell invasion in vitro. The data showed that CAF induced migration of NSCLC A549 and SPCA-1 cells in this three-dimensional invasion microdevice, which is confirmed by using the traditional Transwell system. Furthermore, CAF induced GRP78 expression in A549 and SPCA-1 cells to facilitate NSCLC cell migration and invasion, whereas knockdown of GRP78 expression blocked A549 and SPCA-1 cell migration and invasion capacity. In conclusion, these data indicated that CAFs might promote NSCLC cell invasion by up-regulation of GRP78 expression and this bionic chip microdevice is a robust platform to assess the interaction of cancer and stromal cells in tumor environment study. PMID:27016417

  11. Cancer-associated fibroblasts promote non-small cell lung cancer cell invasion by upregulation of glucose-regulated protein 78 (GRP78) expression in an integrated bionic microfluidic device

    PubMed Central

    Song, Jing; Liu, Yuanbin; Gao, Zhancheng; Wang, Qi

    2016-01-01

    The tumor microenvironment is comprised of cancer cells and various stromal cells and their respective cellular components. Cancer-associated fibroblasts (CAFs), a major part of the stromal cells, are a key determinant in tumor progression, while glucose-regulated protein (GRP)78 is overexpressed in many human cancers and is involved in tumor invasion and metastasis. This study developed a microfluidic-based three dimension (3D) co-culture device to mimic an in vitro tumor microenvironment in order to investigate tumor cell invasion in real-time. This bionic chip provided significant information regarding the role of GRP78, which may be stimulated by CAFs, to promote non-small cell lung cancer cell invasion in vitro. The data showed that CAF induced migration of NSCLC A549 and SPCA-1 cells in this three-dimensional invasion microdevice, which is confirmed by using the traditional Transwell system. Furthermore, CAF induced GRP78 expression in A549 and SPCA-1 cells to facilitate NSCLC cell migration and invasion, whereas knockdown of GRP78 expression blocked A549 and SPCA-1 cell migration and invasion capacity. In conclusion, these data indicated that CAFs might promote NSCLC cell invasion by up-regulation of GRP78 expression and this bionic chip microdevice is a robust platform to assess the interaction of cancer and stromal cells in tumor environment study. PMID:27016417

  12. Cancer-associated fibroblasts promote non-small cell lung cancer cell invasion by upregulation of glucose-regulated protein 78 (GRP78) expression in an integrated bionic microfluidic device.

    PubMed

    Yu, Ting; Guo, Zhe; Fan, Hui; Song, Jing; Liu, Yuanbin; Gao, Zhancheng; Wang, Qi

    2016-05-01

    The tumor microenvironment is comprised of cancer cells and various stromal cells and their respective cellular components. Cancer-associated fibroblasts (CAFs), a major part of the stromal cells, are a key determinant in tumor progression, while glucose-regulated protein (GRP)78 is overexpressed in many human cancers and is involved in tumor invasion and metastasis. This study developed a microfluidic-based three dimension (3D) co-culture device to mimic an in vitro tumor microenvironment in order to investigate tumor cell invasion in real-time. This bionic chip provided significant information regarding the role of GRP78, which may be stimulated by CAFs, to promote non-small cell lung cancer cell invasion in vitro. The data showed that CAF induced migration of NSCLC A549 and SPCA-1 cells in this three-dimensional invasion microdevice, which is confirmed by using the traditional Transwell system. Furthermore, CAF induced GRP78 expression in A549 and SPCA-1 cells to facilitate NSCLC cell migration and invasion, whereas knockdown of GRP78 expression blocked A549 and SPCA-1 cell migration and invasion capacity. In conclusion, these data indicated that CAFs might promote NSCLC cell invasion by up-regulation of GRP78 expression and this bionic chip microdevice is a robust platform to assess the interaction of cancer and stromal cells in tumor environment study.

  13. Light at night alters daily patterns of cortisol and clock proteins in female Siberian hamsters.

    PubMed

    Bedrosian, T A; Galan, A; Vaughn, C A; Weil, Z M; Nelson, R J

    2013-06-01

    Humans and other organisms have adapted to a 24-h solar cycle in response to life on Earth. The rotation of the planet on its axis and its revolution around the sun cause predictable daily and seasonal patterns in day length. To successfully anticipate and adapt to these patterns in the environment, a variety of biological processes oscillate with a daily rhythm of approximately 24 h in length. These rhythms arise from hierarchally-coupled cellular clocks generated by positive and negative transcription factors of core circadian clock gene expression. From these endogenous cellular clocks, overt rhythms in activity and patterns in hormone secretion and other homeostatic processes emerge. These circadian rhythms in physiology and behaviour can be organised by a variety of cues, although they are most potently entrained by light. In recent history, there has been a major change from naturally-occurring light cycles set by the sun, to artificial and sometimes erratic light cycles determined by the use of electric lighting. Virtually every individual living in an industrialised country experiences light at night (LAN) but, despite its prevalence, the biological effects of such unnatural lighting have not been fully considered. Using female Siberian hamsters (Phodopus sungorus), we investigated the effects of chronic nightly exposure to dim light on daily rhythms in locomotor activity, serum cortisol concentrations and brain expression of circadian clock proteins (i.e. PER1, PER2, BMAL1). Although locomotor activity remained entrained to the light cycle, the diurnal fluctuation of cortisol concentrations was blunted and the expression patterns of clock proteins in the suprachiasmatic nucleus and hippocampus were altered. These results demonstrate that chronic exposure to dim LAN can dramatically affect fundamental cellular function and emergent physiology.

  14. Cell research with physically modified microfluidic channels: a review.

    PubMed

    Kim, Sun Min; Lee, Sung Hoon; Suh, Kahp Yang

    2008-07-01

    An overview of the use of physically modified microfluidic channels towards cell research is presented. The physical modification can be realized either by combining embedded physical micro/nanostructures or a topographically patterned substrate at the micro- or nanoscale inside a channel. After a brief description of the background and the importance of the physically modified microfluidic system, various fabrication methods are described based on the materials and geometries of physical structures and channels. Of many operational principles for microfluidics (electrical, magnetic, optical, mechanical, and so on), this review primarily focuses on mechanical operation principles aided by structural modification of the channels. The mechanical forces are classified into (i) hydrodynamic, (ii) gravitational, (iii) capillary, (iv) wetting, and (v) adhesion forces. Throughout this review, we will specify examples where necessary and provide trends and future directions in the field.

  15. Differentially photo-crosslinked polymers enable self-assembling microfluidics

    PubMed Central

    Jamal, Mustapha; Zarafshar, Aasiyeh M.; Gracias, David H.

    2012-01-01

    An important feature of naturally self-assembled systems such as leaves and tissues is that they are curved and have embedded fluidic channels that enable the transport of nutrients to, or removal of waste from, specific three-dimensional (3D) regions. Here, we report the self-assembly of photopatterned polymers, and consequently microfluidic devices, into curved geometries. We discovered that differentially photo-crosslinked SU-8 films spontaneously and reversibly curved upon film de-solvation and re-solvation. Photolithographic patterning of the SU-8 films enabled the self-assembly of cylinders, cubes, and bidirectionally folded sheets. We integrated polydimethylsiloxane (PDMS) microfluidic channels with these SU-8 films to self-assemble curved microfluidic networks. PMID:22068594

  16. Microfluidic Cell Culture Device

    NASA Technical Reports Server (NTRS)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  17. Spatial Manipulation with Microfluidics

    PubMed Central

    Lin, Benjamin; Levchenko, Andre

    2015-01-01

    Biochemical gradients convey information through space, time, and concentration, and are ultimately capable of spatially resolving distinct cellular phenotypes, such as differentiation, proliferation, and migration. How these gradients develop, evolve, and function during development, homeostasis, and various disease states is a subject of intense interest across a variety of disciplines. Microfluidic technologies have become essential tools for investigating gradient sensing in vitro due to their ability to precisely manipulate fluids on demand in well-controlled environments at cellular length scales. This review will highlight their utility for studying gradient sensing along with relevant applications to biology. PMID:25905100

  18. Probing thyroglobulin in undiluted human serum based on pattern recognition and competitive adsorption of proteins

    NASA Astrophysics Data System (ADS)

    Wang, Ran; Huang, Shuai; Li, Jing; Chae, Junseok

    2014-10-01

    Thyroglobulin (Tg) is a sensitive indicator of persistent or recurrent differentiated thyroid cancer of follicular cell origin. Detection of Tg in human serum is challenging as bio-receptors, such as anti-Tg, used in immunoassay have relatively weak binding affinity. We engineer sensing surfaces using the competitive adsorption of proteins, termed the Vroman Effect. Coupled with Surface Plasmon Resonance, the "cross-responsive" interactions of Tg on the engineered surfaces produce uniquely distinguishable multiple signature patterns, which are discriminated using Linear Discriminant Analysis. Tg-spiked samples, down to 2 ng/ml Tg in undiluted human serum, are sensitively and selectively discriminated from the control (undiluted human serum).

  19. Fabrication of polyimide based microfluidic channels for biosensor devices

    NASA Astrophysics Data System (ADS)

    Zulfiqar, Azeem; Pfreundt, Andrea; Svendsen, Winnie Edith; Dimaki, Maria

    2015-03-01

    The ever-increasing complexity of the fabrication process of Point-of-care (POC) devices, due to high demand of functional versatility, compact size and ease-of-use, emphasizes the need of multifunctional materials that can be used to simplify this process. Polymers, currently in use for the fabrication of the often needed microfluidic channels, have limitations in terms of their physicochemical properties. Therefore, the use of a multipurpose biocompatible material with better resistance to the chemical, thermal and electrical environment, along with capability of forming closed channel microfluidics is inevitable. This paper demonstrates a novel technique of fabricating microfluidic devices using polyimide (PI) which fulfills the aforementioned properties criteria. A fabrication process to pattern microfluidic channels, using partially cured PI, has been developed by using a dry etching method. The etching parameters are optimized and compared to those used for fully cured PI. Moreover, the formation of closed microfluidic channel on wafer level by bonding two partially cured PI layers or a partially cured PI to glass with high bond strength has been demonstrated. The reproducibility in uniformity of PI is also compared to the most commonly used SU8 polymer, which is a near UV sensitive epoxy resin. The potential applications of PI processing are POC and biosensor devices integrated with microelectronics.

  20. Fabrication of a Paper-Based Microfluidic Device to Readily Determine Nitrite Ion Concentration by Simple Colorimetric Assay

    ERIC Educational Resources Information Center

    Wang, Bo; Lin, Zhiqiang; Wang, Min

    2015-01-01

    Paper-based microfluidic devices (µPAD) are a burgeoning platform of microfluidic analysis technology. The method described herein is for use in undergraduate and high school chemistry laboratories. A simple and convenient µPAD was fabricated by easy patterning of filter paper using a permanent marker pen. The usefulness of the device was…

  1. Recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices.

    PubMed

    Sasaki, Naoki

    2012-01-01

    AC electrokinetics is a generic term that refers to an induced motion of particles and fluids under nonuniform AC electric fields. The AC electric fields are formed by application of AC voltages to microelectrodes, which can be easily integrated into microfluidic devices by standard microfabrication techniques. Moreover, the magnitude of the motion is large enough to control the mass transfer on the devices. These advantages are attractive for biomolecular analysis on the microfluidic devices, in which the characteristics of small space and microfluidics have been mainly employed. In this review, I describe recent applications of AC electrokinetics in biomolecular analysis on microfluidic devices. The applications include fluid pumping and mixing by AC electrokinetic flow, and manipulation of biomolecules such as DNA and proteins by various AC electrokinetic techniques. Future prospects for highly functional biomolecular analysis on microfluidic devices with the aid of AC electrokinetics are also discussed.

  2. Three-dimensional surface microfluidics enabled by spatiotemporal control of elastic fluidic interface.

    PubMed

    Hong, Lingfei; Pan, Tingrui

    2010-12-01

    As an emerging alternative to the conventional counterpart, surface microfluidics incorporates both intrinsic resistive solid-liquid and elastic frictionless gas-liquid interfaces, leading to unique flow-pressure characteristics. Furthermore, the open-surface microfluidic platforms can be fabricated on a monolithic substrate with high wettability contrast by the previously reported one-step lithographic process of a photosensitive superhydrophobic nanocomposite material, which permits flexible fluidic operations and direct surface modifications. In the paper, we first present three-dimensional microfluidic manipulations utilizing the unconventional gas-liquid interfaces of surface microfluidics, outlined by the micropatterned wetting boundaries (also known as the triple lines). In contrast to the primary linear (resistive) nature of the conventional closed-channel microfluidics, the distinct elastic interface of surface microfluidics enables remarkable three-dimensional (deformable) and time-dependent (capacitive) operations of the flow. Specifically, spatiotemporal dependence of microflow patterns on the planar fluidic surfaces has been theoretically analyzed and experimentally characterized. Utilizing the unconventional interface-enabled flow-pressure relationship, novel surface fluidic operations, including microflow regulation and flow-controlled switching, have been demonstrated and fully investigated. Furthermore, three-dimensional surface microfluidic networks together with analog-to-digital stereo-flow activations have been established, in which miniature capillary bridges form fluidic connections between two independent surface microfluidic circuits.

  3. Fabricating PFPE Membranes for Microfluidic Valves and Pumps

    NASA Technical Reports Server (NTRS)

    Greer, Frank; White, Victor E.; Lee, Michael C.; Willis, Peter A.; Grunthaner, Frank J.; Rolland, Jason; Rolland, Jason

    2009-01-01

    A process has been developed for fabricating membranes of a perfluoropolyether (PFPE) and integrating them into valves and pumps in laboratory-on-achip microfluidic devices. Membranes of poly(tetrafluoroethylene) [PTFE] and poly(dimethylsilane) [PDMS] have been considered for this purpose and found wanting. By making it possible to use PFPE instead of PTFE or PDMS, the present process expands the array of options for further development of microfluidic devices for diverse applications that could include detection of biochemicals of interest, detection of toxins and biowarfare agents, synthesis and analysis of proteins, medical diagnosis, and synthesis of fuels.

  4. Creation of hydrophilic microfluidic devices for biomedical application through stereolithography

    NASA Astrophysics Data System (ADS)

    Brandhoff, Lukas; van den Driesche, Sander; Lucklum, Frieder; Vellekoop, Michael J.

    2015-06-01

    We present a method to graft a layer of poly-ethylene-glycol (PEG) to the surface of stereo-lithography fabricated or 3D-printed microfluidic devices rendering it hydrophilic and repellent to the adhesion of proteins. The PEG forms a rigid bond with the surface that is more stable than many coatings or surface treatments. This makes stereolithography much more attractive as a prototyping platform for microfluidics. The method has been proven with two different resins by different manufacturers, showing the universality of said treatment.

  5. Injury, nerve, and wound epidermis related electrophoretic and fluorographic protein patterns in forelimbs of adult newts

    SciTech Connect

    Garling, D.J.; Tassava, R.A.

    1984-08-01

    Polyacrylamide slab gel electrophoresis and (/sup 35/S)methionine fluorography were used to examine proteins in regenerating newt limbs, amputated denervated limbs, unamputated denervated limbs, and separated blastema mesodermal core and wound epidermis. A total of 27 protein electrophoretic bands were obtained from amputated limbs and 24 bands from unamputated limbs. Amputation resulted in the appearance of 4 new bands and the loss of 1 band as compared to unamputated limbs. These 5 banding differences were apparent on stained gels 3 days postamputation and were maintained through 10 weeks postamputation (complete regenerate stage). Only one band in unamputated limbs was always detectable on fluorographs, whereas virtually all of the stainable bands of amputated limbs were visible on fluorographs. Amputation clearly stimulated a marked, generalized increase in the synthesis of limb proteins. The 5 amputation induced changes were equally evident in stained gels of both innervated and denervated limbs. Amputated denervated limbs possessed a full set of fluorographic bands (including the 5 differences) through 18 days postamputation. However, denervation without amputation was not sufficient to alter the stainable banding pattern. Wound epidermis and mesodermal core both displayed the 5 banding differences and had identical banding patterns with the exception of one epidermal specific band. This band was also present in whole limb skin but was absent in unamputated mesodermal limb tissue. This was the only band of unamputated limbs that was consistently detectable by fluorography. It is concluded that amputation induces nerve independent changes in protein synthesis that are common to both mesodermal core and wound epidermis. These changes may represent preparation for cellular proliferation.

  6. Fabrication of Self-Cleaning, Reusable Titania Templates for Nanometer and Micrometer Scale Protein Patterning.

    PubMed

    Moxey, Mark; Johnson, Alexander; El-Zubir, Osama; Cartron, Michael; Dinachali, Saman Safari; Hunter, C Neil; Saifullah, Mohammad S M; Chong, Karen S L; Leggett, Graham J

    2015-06-23

    The photocatalytic self-cleaning characteristics of titania facilitate the fabrication of reuseable templates for protein nanopatterning. Titania nanostructures were fabricated over square centimeter areas by interferometric lithography (IL) and nanoimprint lithography (NIL). With the use of a Lloyd's mirror two-beam interferometer, self-assembled monolayers of alkylphosphonates adsorbed on the native oxide of a Ti film were patterned by photocatalytic nanolithography. In regions exposed to a maximum in the interferogram, the monolayer was removed by photocatalytic oxidation. In regions exposed to an intensity minimum, the monolayer remained intact. After exposure, the sample was etched in piranha solution to yield Ti nanostructures with widths as small as 30 nm. NIL was performed by using a silicon stamp to imprint a spin-cast film of titanium dioxide resin; after calcination and reactive ion etching, TiO2 nanopillars were formed. For both fabrication techniques, subsequent adsorption of an oligo(ethylene glycol) functionalized trichlorosilane yielded an entirely passive, protein-resistant surface. Near-UV exposure caused removal of this protein-resistant film from the titania regions by photocatalytic degradation, leaving the passivating silane film intact on the silicon dioxide regions. Proteins labeled with fluorescent dyes were adsorbed to the titanium dioxide regions, yielding nanopatterns with bright fluorescence. Subsequent near-UV irradiation of the samples removed the protein from the titanium dioxide nanostructures by photocatalytic degradation facilitating the adsorption of a different protein. The process was repeated multiple times. These simple methods appear to yield durable, reuseable samples that may be of value to laboratories that require nanostructured biological interfaces but do not have access to the infrastructure required for nanofabrication.

  7. Sex hormones and expression pattern of cytoskeletal proteins in the rat brain throughout pregnancy.

    PubMed

    González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; González-Flores, Oscar; Galván-Rosas, Agustín; Porfirio Gómora-Arrati; Camacho-Arroyo, Ignacio

    2014-01-01

    Pregnancy involves diverse changes in brain function that implicate a re-organization in neuronal cytoskeleton. In this physiological state, the brain is in contact with several hormones that it has never been exposed, as well as with very high levels of hormones that the brain has been in touch throughout life. Among the latter hormones are progesterone and estradiol which regulate several brain functions, including learning, memory, neuroprotection, and the display of sexual and maternal behavior. These functions involve changes in the structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity is regulated by estradiol and progesterone. We have found that the expression pattern of Microtubule Associated Protein 2, Tau, and Glial Fibrillary Acidic Protein changes in a tissue-specific manner in the brain of the rat throughout gestation and the start of lactation, suggesting that these proteins participate in the plastic changes observed in the brain during pregnancy. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

  8. Cell patterning via linker-free protein functionalization of an organic conducting polymer (polypyrrole) electrode.

    PubMed

    Bax, Daniel V; Tipa, Roxana S; Kondyurin, Alexey; Higgins, Michael J; Tsoutas, Kostadinos; Gelmi, Amy; Wallace, Gordon G; McKenzie, David R; Weiss, Anthony S; Bilek, Marcela M M

    2012-07-01

    The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of the conducting polymer, polypyrrole, which possesses electrical properties. PIII treatment enabled persistent, covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. In contrast tropoelastin was readily eluted from the untreated surface. Through this differential persistence of binding, surface bound tropoelastin supported cell adhesion and spreading on the PIII treated but not the untreated polypyrrole surface. The application of a steel shadow mask during PIII treatment allowed for spatial definition of tropoelastin exclusively to PIII treated regions. The general applicability of this approach to other extracellular matrix proteins was illustrated using collagen I, which displayed similar results to tropoelastin but required extended washing conditions. This approach allowed fine patterning of cell adhesion and spreading to tropoelastin and collagen, specifically on PIII treated polypyrrole regions. We therefore present a methodology to alter the functionality of polypyrrole surfaces, generating surfaces that can spatially control cellular interactions through protein functionalization with the potential for electrical stimulation.

  9. Active micromachines: Microfluidics powered by mesoscale turbulence.

    PubMed

    Thampi, Sumesh P; Doostmohammadi, Amin; Shendruk, Tyler N; Golestanian, Ramin; Yeomans, Julia M

    2016-07-01

    Dense active matter, from bacterial suspensions and microtubule bundles driven by motor proteins to cellular monolayers and synthetic Janus particles, is characterized by mesoscale turbulence, which is the emergence of chaotic flow structures. By immersing an ordered array of symmetric rotors in an active fluid, we introduce a microfluidic system that exploits spontaneous symmetry breaking in mesoscale turbulence to generate work. The lattice of rotors self-organizes into a spin state where neighboring discs continuously rotate in permanent alternating directions due to combined hydrodynamic and elastic effects. Our virtual prototype demonstrates a new research direction for the design of micromachines powered by the nematohydrodynamic properties of active turbulence.

  10. Active micromachines: Microfluidics powered by mesoscale turbulence

    PubMed Central

    Thampi, Sumesh P.; Doostmohammadi, Amin; Shendruk, Tyler N.; Golestanian, Ramin; Yeomans, Julia M.

    2016-01-01

    Dense active matter, from bacterial suspensions and microtubule bundles driven by motor proteins to cellular monolayers and synthetic Janus particles, is characterized by mesoscale turbulence, which is the emergence of chaotic flow structures. By immersing an ordered array of symmetric rotors in an active fluid, we introduce a microfluidic system that exploits spontaneous symmetry breaking in mesoscale turbulence to generate work. The lattice of rotors self-organizes into a spin state where neighboring discs continuously rotate in permanent alternating directions due to combined hydrodynamic and elastic effects. Our virtual prototype demonstrates a new research direction for the design of micromachines powered by the nematohydrodynamic properties of active turbulence. PMID:27419229

  11. Improving agglutination tests by working in microfluidic channels.

    PubMed

    Degré, G; Brunet, E; Dodge, A; Tabeling, P

    2005-06-01

    Latex agglutination tests are used for the diagnosis of diseases in man and animals. They are generally simple, cheap, and do not require sophisticated equipment, nor highly specialized skills. In this Technical Note, we put latex agglutination tests in a microfluidic format. The experiment is performed in PDMS (polydimethylsiloxane) microchannels, using streptavidin-coated superparamagnetic beads and a magnetic field. The target molecule is biotinylated protein A. By taking full advantage of the microfluidic conditions (scaling down of the detection volume and controlled action of the shear flow), we achieved an analytical sensitivity of 10 fmol l(-1)(several hundreds of fg ml(-1)) and a fast response (a few minutes) ; the test is also quantitative. Performances of agglutination tests can thus be improved by orders of magnitude by adapting them to a microfluidic format; this comes in addition to the usual advantages offered by this technology (integration, high throughput etc.).

  12. Microfluidic chip-based technologies: emerging platforms for cancer diagnosis

    PubMed Central

    2013-01-01

    The development of early and personalized diagnostic protocols is considered the most promising avenue to decrease mortality from cancer and improve outcome. The emerging microfluidic-based analyzing platforms hold high promises to fulfill high-throughput and high-precision screening with reduced equipment cost and low analysis time, as compared to traditional bulky counterparts in bench-top laboratories. This article overviewed the potential applications of microfluidic technologies for detection and monitoring of cancer through nucleic acid and protein biomarker analysis. The implications of the technologies in cancer cytology that can provide functional personalized diagnosis were highlighted. Finally, the future niches for using microfluidic-based systems in tumor screening were briefly discussed. PMID:24070124

  13. Microfluidic Exosome Analysis toward Liquid Biopsy for Cancer.

    PubMed

    He, Mei; Zeng, Yong

    2016-08-01

    Assessment of a tumor's molecular makeup using biofluid samples, known as liquid biopsy, is a prominent research topic in precision medicine for cancer, due to its noninvasive property allowing repeat sampling for monitoring molecular changes of tumors over time. Circulating exosomes recently have been recognized as promising tumor surrogates because they deliver enriched biomarkers, such as proteins, RNAs, and DNA. However, purification and characterization of these exosomes are technically challenging. Microfluidic lab-on-a-chip technology effectively addresses these challenges owing to its inherent advantages in integration and automation of multiple functional modules, enhancing sensing performance, and expediting analysis processes. In this article, we review the state-of-the-art development of microfluidic technologies for exosome isolation and molecular characterization with emphasis on their applications toward liquid biopsy-based analysis of cancer. Finally, we share our perspectives on current challenges and future directions of microfluidic exosome analysis. PMID:27215792

  14. Microfluidic Biochip Design

    NASA Technical Reports Server (NTRS)

    Panzarella, Charles

    2004-01-01

    As humans prepare for the exploration of our solar system, there is a growing need for miniaturized medical and environmental diagnostic devices for use on spacecrafts, especially during long-duration space missions where size and power requirements are critical. In recent years, the biochip (or Lab-on-a-Chip) has emerged as a technology that might be able to satisfy this need. In generic terms, a biochip is a miniaturized microfluidic device analogous to the electronic microchip that ushered in the digital age. It consists of tiny microfluidic channels, pumps and valves that transport small amounts of sample fluids to biosensors that can perform a variety of tests on those fluids in near real time. It has the obvious advantages of being small, lightweight, requiring less sample fluids and reagents and being more sensitive and efficient than larger devices currently in use. Some of the desired space-based applications would be to provide smaller, more robust devices for analyzing blood, saliva and urine and for testing water and food supplies for the presence of harmful contaminants and microorganisms. Our group has undertaken the goal of adapting as well as improving upon current biochip technology for use in long-duration microgravity environments.

  15. Protein immobilization on Ni(II) ion patterns prepared by microcontact printing and dip-pen nanolithography.

    PubMed

    Wu, Chien-Ching; Reinhoudt, David N; Otto, Cees; Velders, Aldrik H; Subramaniam, Vinod

    2010-02-23

    An indirect method of protein patterning by using Ni(II) ion templates for immobilization via a specific metal-protein interaction is described. A nitrilotriacetic acid (NTA)-terminated self-assembled monolayer (SAM) allows oriented binding of histidine-tagged proteins via complexation with late first-row transition metal ions, such as Ni(II). Patterns of nickel(II) ions were prepared on NTA SAM-functionalized glass slides by microcontact printing (microCP) and dip-pen nanolithography (DPN) to obtain micrometer and submicrometer scale patterns. Consecutive dipping of the slides in 6His-protein solutions resulted in the formation of protein patterns, as was subsequently proven by AFM and confocal fluorescence microscopy. This indirect method prevents denaturation of fragile biomolecules caused by direct printing or writing of proteins. Moreover, it yields well-defined patterned monolayers of proteins and, in principle, is indifferent for biomolecules with a high molecular weight. This approach also enabled us to characterize the transfer of Ni(II) ions on fundamental parameters of DPN, such as writing speeds and tip-surface contact times, while writing with the smallest possible ink "molecules" (i.e., metal ions). PMID:20104881

  16. Predicting protein fold pattern with functional domain and sequential evolution information.

    PubMed

    Shen, Hong-Bin; Chou, Kuo-Chen

    2009-02-01

    The fold pattern of a protein is one level deeper than its structural classification, and hence is more challenging and complicated for prediction. Many efforts have been made in this regard, but so far all the reported success rates are still under 70%, indicating that it is extremely difficult to enhance the success rate even by 1% or 2%. To address this problem, here a novel approach is proposed that is featured by combining the functional domain information and the sequential evolution information through a fusion ensemble classifier. The predictor thus developed is called PFP-FunDSeqE. Tests were performed for identifying proteins among their 27 fold patterns. Compared with the existing predictors tested by a same stringent benchmark dataset, the new predictor can, for the first time, achieve over 70% success rate. The PFP-FunDSeqE predictor is freely available to the public as a web server at http://www.csbio.sjtu.edu.cn/bioinf/PFP-FunDSeqE/.

  17. Expression patterns of five polymorphic membrane proteins during the Chlamydia abortus developmental cycle

    PubMed Central

    Wheelhouse, Nick; Sait, Michelle; Wilson, Kim; Aitchison, Kevin; McLean, Kevin; Smith, David G.E.; Longbottom, David

    2012-01-01

    It has been suggested that polymorphic membrane proteins (Pmps) belonging to the Type V autotransporter protein family play an important role in the pathogenesis of Chlamydia abortus (C. abortus; formerly Chlamydophila abortus) infection. In a previous study we demonstrated the expression of all the pmps at the transcriptional level. The purpose of this study was to measure the number of Pmp positive inclusions throughout the C. abortus developmental cycle to investigate heterogeneity in expression patterns. McCoy cells were infected with C. abortus and analysed for Pmp expression over a 72 h period by fluorescent immunocytochemistry. Pmp18D could be detected at all analysed time points, and could only be accurately quantified from 36 hpi while Pmp10G positive inclusions could be visualised from 36 hpi. Expression of Pmps 13G, 16G and 17G could only be visualised later in the cycle and within less than half of visualised inclusions. These results indicate that while expression of specific Pmps is constitutive (Pmp18D), the pattern of expression of other Pmps is more variable. This suggests that different members of the Pmp family may play different roles within the developmental cycle of the organism, with some (Pmps10G and 18D) having roles throughout the cycle, while the heterogeneity of expression of others may aid in antigenic variation. PMID:22776512

  18. A fibrinogen-related protein identified from hepatopancreas of crayfish is a potential pattern recognition receptor.

    PubMed

    Chen, Qiming; Bai, Suhua; Dong, Chaohua

    2016-09-01

    Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A. hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A. hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor. PMID:27417229

  19. Adaptation of Salmonella enterica Hadar under static magnetic field: effects on outer membrane protein pattern

    PubMed Central

    2012-01-01

    Background Salmonella enterica serovar Hadar (S. Hadar) is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments. Results The present study investigated the adaptation of S. Hadar under the effect of acute static magnetic field exposure (200 mT, 9 h) and the impact on the outer membrane protein pattern. Via two-dimensional electrophoresis (2-DE) and LC-MS/MS spectrometry, we compared the proteome of enriched-outer membrane fraction before and after exposure to a magnetic field. A total of 11 proteins, displaying more than a two-fold change, were differentially expressed in exposed cells, among which 7 were up-regulated and 4 down-regulated. These proteins were involved in the integrity of cell envelope (TolB, Pal), in the response to oxidative stress (OmpW, dihydrolipoamide dehydrogenase, UspF), in the oxidative stress status (bacterioferritin), in virulence (OmpX, Yfgl) or in motility (FlgE and UspF). Complementary experiments associated the down-regulation of FlgE and UspF with an alteration of swarming, a flagella-driven motility, under SMF. Furthermore, the antibiotic disc diffusion method confirmed a decrease of gentamicin susceptibility in exposed cells. This decrease could be partly associated with the up-regulation of TolC, outer membrane component of an efflux pump. OmpA, a multifunctional protein, was up-regulated. Conclusions SMF (200 mT) seems to maintain the cell envelope integrity and to submit the exposed cells to an oxidative stress. Some alterations suggest an increase of the ability of exposed cells to form biofilms. PMID:22304719

  20. Pattern of mineralization after regenerative periodontal therapy with enamel matrix proteins.

    PubMed

    Bosshardt, Dieter D; Sculean, Anton; Donos, Nikolaos; Lang, Niklaus P

    2006-05-01

    A derivative (EMD) of enamel matrix proteins (EMPs) is used for periodontal regeneration because EMPs are believed to induce the formation of acellular extrinsic fiber cementum (AEFC). Other reports, however, indicate that EMPs have osteogenic potential. The aim of this study was to characterize the nature of the tissue that forms on the root surface following application of EMD. Ten human teeth affected by periodontitis and scheduled for extraction were treated with EMD. Four to six weeks later, they were extracted and processed for analysis by light microscopy and transmission electron microscopy. Immunocytochemistry with antibodies against bone sialoprotein (BSP) and osteopontin (OPN) was performed to determine the mineralization pattern. The newly formed tissues on the root were thick and contained embedded cells. Small mineralization foci were regularly seen, and large organic matrix patches were occasionally seen, but a distinct mineralization front was lacking. While labeling for BSP was always associated with small mineralization foci and large matrix patches, OPN labeling was seen inconsistently. It is concluded that tissues resembling either cellular intrinsic fiber cementum or a type of bone were observed. The mineralization pattern mostly resembled that found in bone, except for a few areas that exhibited a hitherto undescribed mineralization pattern. PMID:16674690

  1. XFINGER: a tool for searching and visualising protein fingerprints and patterns.

    PubMed

    Perkins, D N; Attwood, T K

    1996-04-01

    A tool for searching pattern and fingerprint databases is described. Fingerprints are groups of motifs excised from conserved regions of sequence alignments and used for iterative database scanning. The constituent motifs are thus encoded as small alignments in which sequence information is maximised with each database pass; they therefore differ from regular-expression patterns, in which alignments are reduced to single consensus sequences. Different database formats have evolved to store these disparate types of information, namely the PROSITE dictionary of patterns and the PRINTS fingerprint database, but programs have not been available with the flexibility to search them both. We have developed a facility to do this: the system allows query sequences to be scanned against either PROSITE, the full PRINTS database, or against individual fingerprints. The results of fingerprint searches are displayed simultaneously in both text and graphical windows to render them more tangible to the user. Where structural coordinates are available, identified motifs may be visualised in a 3D context. The program runs on Silicon Graphics machines using GL graphics libraries and on machines with X servers supporting the PEX extension: its use is illustrated here by depicting the location of low-density lipoprotein-binding (LDL) motifs and leucine-rich repeats in a mosaic G-protein-coupled receptor (GPCR).

  2. A Droplet Microfluidic Platform for Automating Genetic Engineering.

    PubMed

    Gach, Philip C; Shih, Steve C C; Sustarich, Jess; Keasling, Jay D; Hillson, Nathan J; Adams, Paul D; Singh, Anup K

    2016-05-20

    We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.

  3. A Droplet Microfluidic Platform for Automating Genetic Engineering.

    PubMed

    Gach, Philip C; Shih, Steve C C; Sustarich, Jess; Keasling, Jay D; Hillson, Nathan J; Adams, Paul D; Singh, Anup K

    2016-05-20

    We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput. PMID:26830031

  4. Superporous agarose beads as a solid support for microfluidic immunoassay.

    PubMed

    Yang, Yoonsun; Nam, Seong-Won; Lee, Nae Yoon; Kim, Youn Sang; Park, Sungsu

    2008-09-01

    We demonstrate here with the feasibility of superporous agarose (SA) beads as a solid support in microfluidic immunoassay by detecting goat IgG. In our procedure, SA beads containing superpores were covalently conjugated to protein A. The conjugated beads were introduced into a polydimethyl siloxane microfluidic device. The sandwich immunoassay was performed in the microfluidic device by subsequently introducing anti-goat IgG as the primary antibodies, goat IgG as analytes, alkaline phosphatase-conjugated F(ab')2 anti-goat IgG as detection antibodies, and 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium as substrate in a flow. Depending on the goat IgG concentration, dark and pinky precipitates appeared inside the microchannel immediately after the introduction of all the reagents. The minimum detection limit, 100 pg goat IgG/mL in PBS, was achieved with the naked eye. This enhanced sensitivity is mainly because analytical reagents were allowed to access the outer surface as well as the inner matrices of the beads. This is supported by the facts that the binding of fluorescein isothiocyanate IgG happened throughout the inside matrices of protein A-conjugated SA beads but was limited to the outer surface of protein A-conjugated homogeneous agarose beads. These results suggest that SA beads are highly suitable as a solid support for microfluidic immunoassays.

  5. A Robust and Engineerable Self-Assembling Protein Template for the Synthesis and Patterning of Ordered Nanoparticle Arrays

    NASA Technical Reports Server (NTRS)

    McMillan, R. Andrew; Howard, Jeanie; Zaluzec, Nestor J.; Kagawa, Hiromi K.; Li, Yi-Fen; Paavola, Chad D.; Trent, Jonathan D.

    2004-01-01

    Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here we introduce a general technique for patterning materials on the nanoscale using genetically modified protein cage structures called chaperonins that self-assemble into crystalline templates. Constrained chemical synthesis of transition metal nanoparticles is specific to templates genetically functionalized with poly-Histidine sequences. These arrays of materials are ordered by the nanoscale structure of the crystallized protein. This system may be easily adapted to pattern a variety of materials given the rapidly growing list of peptide sequences selected by screening for specificity for inorganic materials.

  6. Expression pattern analysis of odorant-binding proteins in the pea aphid Acyrthosiphon pisum.

    PubMed

    De Biasio, Filomena; Riviello, Lea; Bruno, Daniele; Grimaldi, Annalisa; Congiu, Terenzio; Sun, Yu Feng; Falabella, Patrizia

    2015-04-01

    Odorant-binding proteins (OBPs) are soluble proteins mediating chemoreception in insects. In previous research, we investigated the molecular mechanisms adopted by aphids to detect the alarm pheromone (E)-β-farnesene and we found that the recognition of this and structurally related molecules is mediated by OBP3 and OBP7. Here, we show the differential expression patterns of 5 selected OBPs (OBP1, OBP3, OBP6, OBP7, OBP8) obtained performing quantitative RT-PCR and immunolocalization experiments in different body parts of adults and in the 5 developmental instars, including winged and unwinged morphs, of the pea aphid Acyrthosiphon pisum. The results provide an overall picture that allows us to speculate on the relationship between the differential expression of OBPs and their putative function. The expression of OBP3, OBP6, and OBP7 in the antennal sensilla suggests a chemosensory function for these proteins, whereas the constant expression level of OBP8 in all instars could suggest a conserved role. Moreover, OBP1 and OBP3 are also expressed in nonsensory organs. A light and scanning electron microscopy study of sensilla on different body parts of aphid, in particular antennae, legs, mouthparts, and cornicles-cauda, completes this research providing a guide to facilitate the mapping of OBP expression profiles.

  7. Zebrafish Thsd7a is a neural protein required for angiogenic patterning during development.

    PubMed

    Wang, Chieh-Huei; Chen, I-Hui; Kuo, Meng-Wei; Su, Pei-Tsu; Lai, Zih-Yin; Wang, Chian-Huei; Huang, Wei-Chang; Hoffman, Jana; Kuo, Calvin J; You, May-Su; Chuang, Yung-Jen

    2011-06-01

    Angiogenesis is a highly organized process under the control of guidance cues that direct endothelial cell (EC) migration. Recently, many molecules that were initially described as regulators of neural guidance were subsequently shown to also direct EC migration. Here, we report a novel protein, thrombospondin type I domain containing 7A (Thsd7a), that is a neural molecule required for directed EC migration during embryonic angiogenesis in zebrafish. Thsd7a is a vertebrate conserved protein. Zebrafish thsd7a transcript was detected along the ventral edge of the neural tube in the developing zebrafish embryos, correlating with the growth path of angiogenic intersegmental vessels (ISVs). Morpholino-knockdown of Thsd7a caused a lateral deviation of angiogenic ECs below the thsd7a-expressing sites, resulting in aberrant ISV patterning. Collectively, our study shows that zebrafish Thsd7a is a neural protein required for ISV angiogenesis, and suggests an important role of Thsd7a in the neurovascular interaction during zebrafish development.

  8. Altered temporal patterns of anxiety in aged and amyloid precursor protein (APP) transgenic mice

    PubMed Central

    Bedrosian, Tracy A.; Herring, Kamillya L.; Weil, Zachary M.; Nelson, Randy J.

    2011-01-01

    Both normal aging and dementia are associated with dysregulation of the biological clock, which contributes to disrupted circadian organization of physiology and behavior. Diminished circadian organization in conjunction with the loss of cholinergic input to the cortex likely contributes to impaired cognition and behavior. One especially notable and relatively common circadian disturbance among the aged is “sundowning syndrome,” which is characterized by exacerbated anxiety, agitation, locomotor activity, and delirium during the hours before bedtime. Sundowning has been reported in both dementia patients and cognitively intact elderly individuals living in institutions; however, little is known about temporal patterns in anxiety and agitation, and the neurobiological basis of these rhythms remains unspecified. In the present study, we explored the diurnal pattern of anxiety-like behavior in aged and amyloid precursor protein (APP) transgenic mice. We then attempted to treat the observed behavioral disturbances in the aged mice using chronic nightly melatonin treatment. Finally, we tested the hypothesis that time-of-day differences in acetylcholinesterase and choline acetyltransferase expression and general neuronal activation (i.e., c-Fos expression) coincide with the behavioral symptoms. Our results show a temporal pattern of anxiety-like behavior that emerges in elderly mice. This behavioral pattern coincides with elevated locomotor activity relative to adult mice near the end of the dark phase, and with time-dependent changes in basal forebrain acetylcholinesterase expression. Transgenic APP mice show a similar behavioral phenomenon that is not observed among age-matched wild-type mice. These results may have useful applications to the study and treatment of age- and dementia-related circadian behavioral disturbances, namely, sundowning syndrome. PMID:21709248

  9. Solvent resistant microfluidic DNA synthesizer.

    PubMed

    Huang, Yanyi; Castrataro, Piero; Lee, Cheng-Chung; Quake, Stephen R

    2007-01-01

    We fabricated a microfluidic DNA synthesizer out of perfluoropolyether (PFPE), an elastomer with excellent chemical compatibility which makes it possible to perform organic chemical reactions, and synthesized 20-mer oligonucleotides on chip. PMID:17180201

  10. Passive microfluidic array card and reader

    SciTech Connect

    Dugan, Lawrence Christopher; Coleman, Matthew A.

    2011-08-09

    A microfluidic array card and reader system for analyzing a sample. The microfluidic array card includes a sample loading section for loading the sample onto the microfluidic array card, a multiplicity of array windows, and a transport section or sections for transporting the sample from the sample loading section to the array windows. The microfluidic array card reader includes a housing, a receiving section for receiving the microfluidic array card, a viewing section, and a light source that directs light to the array window of the microfluidic array card and to the viewing section.

  11. Towards printable open air microfluidics.

    SciTech Connect

    Collord, Andrew; Cook, Adam W.; Clem, Paul Gilbert; Fenton, Kyle Ross; Apblett, Christopher Alan; Branson, Eric D.

    2010-04-01

    We have demonstrated a novel microfluidic technique for aqueous media, which uses super-hydrophobic materials to create microfluidic channels that are open to the atmosphere. We have demonstrated the ability to perform traditional electrokinetic operations such as ionic separations and electrophoresis using these devices. The rate of evaporation was studied and found to increase with decreasing channel size, which places a limitation on the minimum size of channel that could be used for such a device.

  12. [Identification and expression patterns of anterior silk gland specific cuticle protein Bm11721 in the silkworm (Bombyx mori)].

    PubMed

    Xie, Kang; Wang, Xin; Chen, Huifang; Li, Yi; Song, Qianru; Zhao, Ping

    2016-01-01

    The silk gland of silkworm is the organ of silk protein synthesis and secretion. According to the morphological and functional differences, silk gland can be divided into anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). ASG is the place for silk proteins conformation changes although it cannot synthetize silk proteins. ASG has narrow luminal structures and rigid wall which consists of chitin and cuticle proteins so that it can provide the shearing force which plays an important role in the silk protein conformation changes. The objective of this study is to identify the new chitin binding proteins in ASG of silkworm (Bombyx mori), and to analyze their expression patterns in different tissues. We identified a cuticle protein with chitin binding domain Bml1721 (GenBank Accession No. NM-001173285.1) by chitin affinity chromatography column. We also expressed the recombinant protein as inclusion body using the prokaryotic expression system, and then successfully purified the recombinant protein by nickel affinity chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bm11721 was specifically expressed in ASG. Furthermore, the expression level of Bm 11721 protein was unchanged during the 5th instar. Immunofluorescence analysis revealed that Bm1 1721 was located in the ASG inner membrane. It is proposed that Bm11721 is a component of inner membrane and probably provides the shearing force for conformational changes.

  13. [Identification and expression patterns of anterior silk gland specific cuticle protein Bm11721 in the silkworm (Bombyx mori)].

    PubMed

    Xie, Kang; Wang, Xin; Chen, Huifang; Li, Yi; Song, Qianru; Zhao, Ping

    2016-01-01

    The silk gland of silkworm is the organ of silk protein synthesis and secretion. According to the morphological and functional differences, silk gland can be divided into anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). ASG is the place for silk proteins conformation changes although it cannot synthetize silk proteins. ASG has narrow luminal structures and rigid wall which consists of chitin and cuticle proteins so that it can provide the shearing force which plays an important role in the silk protein conformation changes. The objective of this study is to identify the new chitin binding proteins in ASG of silkworm (Bombyx mori), and to analyze their expression patterns in different tissues. We identified a cuticle protein with chitin binding domain Bml1721 (GenBank Accession No. NM-001173285.1) by chitin affinity chromatography column. We also expressed the recombinant protein as inclusion body using the prokaryotic expression system, and then successfully purified the recombinant protein by nickel affinity chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bm11721 was specifically expressed in ASG. Furthermore, the expression level of Bm 11721 protein was unchanged during the 5th instar. Immunofluorescence analysis revealed that Bm1 1721 was located in the ASG inner membrane. It is proposed that Bm11721 is a component of inner membrane and probably provides the shearing force for conformational changes. PMID:27363199

  14. Pen microfluidics: rapid desktop manufacturing of sealed thermoplastic microchannels.

    PubMed

    Rahmanian, Omid; DeVoe, Don L

    2013-03-21

    A unique technique for the rapid fabrication of thermoplastic microfluidic chips is described. The method enables the realization of fully-sealed microchannels in around one hour while requiring only minimal infrastructure by taking advantage of a solvent swelling mechanism that allows raised features to be patterned on the surface of homogeneous thermoplastic materials. Patterning is achieved without photolithography by simply drawing the desired microchannel pattern onto the polymer surface using a suitable ink as a masking layer, either manually or under robotic control, followed by timed exposure to solvent vapor to yield a desired depth for the masked channel features. The channels are then permanently sealed through solvent bonding of the microchannel chip to a mating thermoplastic substrate. The process is demonstrated using cyclic olefin copolymer as a thermoplastic material, with fully operational microfluidic devices fabricated following a true desktop manufacturing model suitable for rapid prototyping. PMID:23344819

  15. Pen microfluidics: rapid desktop manufacturing of sealed thermoplastic microchannels.

    PubMed

    Rahmanian, Omid; DeVoe, Don L

    2013-03-21

    A unique technique for the rapid fabrication of thermoplastic microfluidic chips is described. The method enables the realization of fully-sealed microchannels in around one hour while requiring only minimal infrastructure by taking advantage of a solvent swelling mechanism that allows raised features to be patterned on the surface of homogeneous thermoplastic materials. Patterning is achieved without photolithography by simply drawing the desired microchannel pattern onto the polymer surface using a suitable ink as a masking layer, either manually or under robotic control, followed by timed exposure to solvent vapor to yield a desired depth for the masked channel features. The channels are then permanently sealed through solvent bonding of the microchannel chip to a mating thermoplastic substrate. The process is demonstrated using cyclic olefin copolymer as a thermoplastic material, with fully operational microfluidic devices fabricated following a true desktop manufacturing model suitable for rapid prototyping.

  16. Pen microfluidics: rapid desktop manufacturing of sealed thermoplastic microchannels

    PubMed Central

    Rahmanian, Omid

    2013-01-01

    A unique technique for the rapid fabrication of thermoplastic microfluidic chips is described. The method enables the realization of fully-sealed microchannels in around one hour while requiring only minimal infrastructure by taking advantage of a solvent swelling mechanism that allows raised features to be patterned on the surface of homogeneous thermoplastic materials. Patterning is achieved without photolithography by simply drawing the desired microchannel pattern onto the polymer surface using a suitable ink as a masking layer, either manually or under robotic control, followed by timed exposure to solvent vapor to yield a desired depth for the masked channel features. The channels are then permanently sealed through solvent bonding of the microchannel chip to a mating thermoplastic substrate. The process is demonstrated using cyclic olefin copolymer as a thermoplastic material, with fully operational microfluidic devices fabricated following a true desktop manufacturing model suitable for rapid prototyping. PMID:23344819

  17. Automated Learning of Subcellular Variation among Punctate Protein Patterns and a Generative Model of Their Relation to Microtubules

    PubMed Central

    Johnson, Gregory R.; Li, Jieyue; Shariff, Aabid; Rohde, Gustavo K.; Murphy, Robert F.

    2015-01-01

    Characterizing the spatial distribution of proteins directly from microscopy images is a difficult problem with numerous applications in cell biology (e.g. identifying motor-related proteins) and clinical research (e.g. identification of cancer biomarkers). Here we describe the design of a system that provides automated analysis of punctate protein patterns in microscope images, including quantification of their relationships to microtubules. We constructed the system using confocal immunofluorescence microscopy images from the Human Protein Atlas project for 11 punctate proteins in three cultured cell lines. These proteins have previously been characterized as being primarily located in punctate structures, but their images had all been annotated by visual examination as being simply “vesicular”. We were able to show that these patterns could be distinguished from each other with high accuracy, and we were able to assign to one of these subclasses hundreds of proteins whose subcellular localization had not previously been well defined. In addition to providing these novel annotations, we built a generative approach to modeling of punctate distributions that captures the essential characteristics of the distinct patterns. Such models are expected to be valuable for representing and summarizing each pattern and for constructing systems biology simulations of cell behaviors. PMID:26624011

  18. Plasma stencilling methods for cell patterning.

    PubMed

    Frimat, Jean-Philippe; Menne, Heike; Michels, Antje; Kittel, Silke; Kettler, Raffael; Borgmann, Sabine; Franzke, Joachim; West, Jonathan

    2009-10-01

    In this paper we describe plasma stencilling techniques for patterning 10 mammalian cell lines on hydrophobic and cell repellent poly(dimethylsiloxane) (PDMS), methylated glass and bacterial grade polystyrene surfaces. An air plasma produced with a Tesla generator operating at atmospheric pressure was used with microengineered stencils for patterned surface oxidation, selectively transforming the surface to a hydrophilic state to enable cell adhesion and growth. Plasma stencilling obviates the need for directly patterning cell adhesion molecules. Instead, during cell culture, adhesion proteins from the media assemble in a bioactive form on the hydrophilic regions. Critically, the removal of protein patterning prior to cell culture provides the option to also use PDMS-PDMS plasma bonding to incorporate cell patterns within microfluidic systems. Linear patterns were generated using PDMS microchannel stencils, and polyimide stencils with through holes were used for the production of cellular arrays. For the production of smaller cellular arrays, a novel microcapillary-based dielectric barrier discharge system was developed. A numerical method to characterise the cell patterns is also introduced and was used to demonstrate that plasma stencilling is highly effective, with complete patterns confined during long term cell culture (>10 days). In summary, plasma stencilling is simple, rapid, inexpensive, reproducible and a potentially universal cell line patterning capability.

  19. The application of counter immunoelectrophoresis (CIE) in ocular protein studies. Part I: time dependent deposition patterns of immunoregulatory proteins on anionic hydrogel contact lenses.

    PubMed

    Mann, Aisling M; Jones, Lyndon W; Tighe, Brian J

    2002-06-01

    This paper focuses on the effects of wear regime on the deposition pattern of important immunoregulatory proteins on FDA Group IV etafilcon-A lenses. Specifically, the aim was to assess the extent to which the daily disposable wear modality produces a different deposition of proteins from the conventional daily wear regime which is coupled with cleaning and disinfection. Counter immunoelectrophoresis (CIE) was employed to detect individual proteins in lens extracts from individual patients and focused on the analysis of five proteins, IgA, IgG, lactoferrin, albumin and kininogen. Deposition was monitored as a function of time; significantly lower deposition was detected on the daily disposable lenses.

  20. Two different immunostaining patterns of beta-amyloid precursor protein (APP) may distinguish traumatic from nontraumatic axonal injury.

    PubMed

    Hayashi, Takahito; Ago, Kazutoshi; Nakamae, Takuma; Higo, Eri; Ogata, Mamoru

    2015-09-01

    Immunostaining for beta-amyloid precursor protein (APP) is recognized as an effective tool for detecting traumatic axonal injury, but it also detects axonal injury due to ischemic or other metabolic causes. Previously, we reported two different patterns of APP staining: labeled axons oriented along with white matter bundles (pattern 1) and labeled axons scattered irregularly (pattern 2) (Hayashi et al. (Leg Med (Tokyo) 11:S171-173, 2009). In this study, we investigated whether these two patterns are consistent with patterns of trauma and hypoxic brain damage, respectively. Sections of the corpus callosum from 44 cases of blunt head injury and equivalent control tissue were immunostained for APP. APP was detected in injured axons such as axonal bulbs and varicose axons in 24 of the 44 cases of head injuries that also survived for three or more hours after injury. In 21 of the 24 APP-positive cases, pattern 1 alone was observed in 14 cases, pattern 2 alone was not observed in any cases, and both patterns 1 and 2 were detected in 7 cases. APP-labeled injured axons were detected in 3 of the 44 control cases, all of which were pattern 2. These results suggest that pattern 1 indicates traumatic axonal injury, while pattern 2 results from hypoxic insult. These patterns may be useful to differentiate between traumatic and nontraumatic axonal injuries. PMID:26249371

  1. Two different immunostaining patterns of beta-amyloid precursor protein (APP) may distinguish traumatic from nontraumatic axonal injury.

    PubMed

    Hayashi, Takahito; Ago, Kazutoshi; Nakamae, Takuma; Higo, Eri; Ogata, Mamoru

    2015-09-01

    Immunostaining for beta-amyloid precursor protein (APP) is recognized as an effective tool for detecting traumatic axonal injury, but it also detects axonal injury due to ischemic or other metabolic causes. Previously, we reported two different patterns of APP staining: labeled axons oriented along with white matter bundles (pattern 1) and labeled axons scattered irregularly (pattern 2) (Hayashi et al. (Leg Med (Tokyo) 11:S171-173, 2009). In this study, we investigated whether these two patterns are consistent with patterns of trauma and hypoxic brain damage, respectively. Sections of the corpus callosum from 44 cases of blunt head injury and equivalent control tissue were immunostained for APP. APP was detected in injured axons such as axonal bulbs and varicose axons in 24 of the 44 cases of head injuries that also survived for three or more hours after injury. In 21 of the 24 APP-positive cases, pattern 1 alone was observed in 14 cases, pattern 2 alone was not observed in any cases, and both patterns 1 and 2 were detected in 7 cases. APP-labeled injured axons were detected in 3 of the 44 control cases, all of which were pattern 2. These results suggest that pattern 1 indicates traumatic axonal injury, while pattern 2 results from hypoxic insult. These patterns may be useful to differentiate between traumatic and nontraumatic axonal injuries.

  2. Microfluidic serial dilution ladder.

    PubMed

    Ahrar, Siavash; Hwang, Michelle; Duncan, Philip N; Hui, Elliot E

    2014-01-01

    Serial dilution is a fundamental procedure that is common to a large number of laboratory protocols. Automation of serial dilution is thus a valuable component for lab-on-a-chip systems. While a handful of different microfluidic strategies for serial dilution have been reported, approaches based on continuous flow mixing inherently consume larger amounts of sample volume and chip real estate. We employ valve-driven circulatory mixing to address these issues and also introduce a novel device structure to store each stage of the dilution process. The dilution strategy is based on sequentially mixing the rungs of a ladder structure. We demonstrate a 7-stage series of 1 : 1 dilutions with R(2) equal to 0.995 in an active device area of 1 cm(2).

  3. Enhanced Microfluidic Electromagnetic Measurements

    NASA Technical Reports Server (NTRS)

    Giovangrandi, Laurent (Inventor); Ricco, Antonio J. (Inventor); Kovacs, Gregory (Inventor)

    2015-01-01

    Techniques for enhanced microfluidic impedance spectroscopy include causing a core fluid to flow into a channel between two sheath flows of one or more sheath fluids different from the core fluid. Flow in the channel is laminar. A dielectric constant of a fluid constituting either sheath flow is much less than a dielectric constant of the core fluid. Electrical impedance is measured in the channel between at least a first pair of electrodes. In some embodiments, enhanced optical measurements include causing a core fluid to flow into a channel between two sheath flows of one or more sheath fluids different from the core fluid. An optical index of refraction of a fluid constituting either sheath flow is much less than an optical index of refraction of the core fluid. An optical property is measured in the channel.

  4. Inertial Focusing in Microfluidics

    PubMed Central

    Martel, Joseph M.; Toner, Mehmet

    2015-01-01

    When Segré and Silberberg in 1961 witnessed particles in a laminar pipe flow congregating at an annulus in the pipe, scientists were perplexed and spent decades learning why such behavior occurred, finally understanding that it was caused by previously unknown forces on particles in an inertial flow. The advent of microfluidics opened a new realm of possibilities for inertial focusing in the processing of biological fluids and cellular suspensions and created a field that is now rapidly expanding. Over the past five years, inertial focusing has enabled high-throughput, simple, and precise manipulation of bodily fluids for a myriad of applications in point-of-care and clinical diagnostics. This review describes the theoretical developments that have made the field of inertial focusing what it is today and presents the key applications that will make inertial focusing a mainstream technology in the future. PMID:24905880

  5. Electrodes for microfluidic applications

    DOEpatents

    Crocker, Robert W.; Harnett, Cindy K.; Rognlien, Judith L.

    2006-08-22

    An electrode device for high pressure applications. These electrodes, designed to withstand pressure of greater than 10,000 psi, are adapted for use in microfluidic devices that employ electrokinetic or electrophoretic flow. The electrode is composed, generally, of an outer electrically insulating tubular body having a porous ceramic frit material disposed in one end of the outer body. The pores of the porous ceramic material are filled with an ion conductive polymer resin. A conductive material situated on the upper surface of the porous ceramic frit material and, thus isolated from direct contact with the electrolyte, forms a gas diffusion electrode. A metal current collector, in contact with the gas diffusion electrode, provides connection to a voltage source.

  6. Digital Microfluidic Cell Culture.

    PubMed

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF. PMID:26643019

  7. One-step two-dimensional microfluidics-based synthesis of three-dimensional particles.

    PubMed

    Hakimi, Navid; Tsai, Scott S H; Cheng, Chil-Hung; Hwang, Dae Kun

    2014-03-01

    Synthesis of three-dimensional anisotropic microparticles using a simple one-step microfluidic-based method is described. The method exploits the nonuniformity of the polymerizing UV light, UV absorption by opaque nanoparticles in the precursor solution, and discontinuous photomask patterns to make magnetic and non-magnetic microparticles in a twodimensional microchannel. Numerical simulations of monomer conversion in the microfluidic channel are performed to predict the manufactured particle shape. PMID:24327458

  8. Accelerating Yeast Prion Biology using Droplet Microfluidics

    NASA Astrophysics Data System (ADS)

    Ung, Lloyd; Rotem, Assaf; Jarosz, Daniel; Datta, Manoshi; Lindquist, Susan; Weitz, David

    2012-02-01

    Prions are infectious proteins in a misfolded form, that can induce normal proteins to take the misfolded state. Yeast prions are relevant, as a model of human prion diseases, and interesting from an evolutionary standpoint. Prions may also be a form of epigenetic inheritance, which allow yeast to adapt to stressful conditions at rates exceeding those of random mutations and propagate that adaptation to their offspring. Encapsulation of yeast in droplet microfluidic devices enables high-throughput measurements with single cell resolution, which would not be feasible using bulk methods. Millions of populations of yeast can be screened to obtain reliable measurements of prion induction and loss rates. The population dynamics of clonal yeast, when a fraction of the cells are prion expressing, can be elucidated. Furthermore, the mechanism by which certain strains of bacteria induce yeast to express prions in the wild can be deduced. Integrating the disparate fields of prion biology and droplet microfluidics reveals a more complete picture of how prions may be more than just diseases and play a functional role in yeast.

  9. Polaris, a protein involved in left-right axis patterning, localizes to basal bodies and cilia.

    PubMed

    Taulman, P D; Haycraft, C J; Balkovetz, D F; Yoder, B K

    2001-03-01

    Mutations in Tg737 cause a wide spectrum of phenotypes, including random left-right axis specification, polycystic kidney disease, liver and pancreatic defects, hydrocephalus, and skeletal patterning abnormalities. To further assess the biological function of Tg737 and its role in the mutant pathology, we identified the cell population expressing Tg737 and determined the subcellular localization of its protein product called Polaris. Tg737 expression is associated with cells possessing either motile or immotile cilia and sperm. Similarly, Polaris concentrated just below the apical membrane in the region of the basal bodies and within the cilia or flagellar axoneme. The data suggest that Polaris functions in a ciliogenic pathway or in cilia maintenance, a role supported by the loss of cilia on the ependymal cell layer in ventricles of Tg737(orpk) brains and by the lack of node cilia in Tg737(Delta2-3betaGal) mutants. PMID:11251073

  10. Microfluidic self-assembly of insulin monomers into amyloid fibrils on a solid surface.

    PubMed

    Lee, Joon Seok; Um, Eujin; Park, Je-Kyun; Park, Chan Beum

    2008-07-15

    We report the self-assembly of insulin monomers into amyloid fibrils within microchannels. To demonstrate the microfluidic amyloid formation and fibril growth on a solid surface, we seeded the internal surfaces of the microchannels with insulin monomers via N-hydroxysuccinimide ester activation and continuously flushed a fresh insulin solution through the microchannels. According to our analysis using optical and fluorescence microscopy, insulin amyloid preferentially formed in the center of the microchannels and, after reaching a certain density, spread to the side walls of the microchannels. By using ex situ atomic force microscopy, we observed the growth of amyloid fibrils inside the microchannels, which occurred at a much higher rate than that in bulk systems. After 12 h of incubation, insulin formed amyloid spherulites having "Maltese cross" extinction patterns within the microchannels according to the polarized microscopic analysis. Microfluidic amyloid formation enabled low consumption of reagents, reduction of incubation time, and simultaneous observation of amyloid formation under different conditions. This work will contribute to the rapid analysis of amyloid formation associated with many protein misfolding diseases.

  11. Droplet Microfluidics for Chip-Based Diagnostics

    PubMed Central

    Kaler, Karan V. I. S.; Prakash, Ravi

    2014-01-01

    Droplet microfluidics (DMF) is a fluidic handling technology that enables precision control over dispensing and subsequent manipulation of droplets in the volume range of microliters to picoliters, on a micro-fabricated device. There are several different droplet actuation methods, all of which can generate external stimuli, to either actively or passively control the shape and positioning of fluidic droplets over patterned substrates. In this review article, we focus on the operation and utility of electro-actuation-based DMF devices, which utilize one or more micro-/nano-patterned substrates to facilitate electric field-based handling of chemical and/or biological samples. The underlying theory of DMF actuations, device fabrication methods and integration of optical and opto-electronic detectors is discussed in this review. Example applications of such electro-actuation-based DMF devices have also been included, illustrating the various actuation methods and their utility in conducting chip-based laboratory and clinical diagnostic assays. PMID:25490590

  12. Metabolic viability of Escherichia coli trapped by dielectrophoresis in microfluidics.

    PubMed

    Donato, Sandra S; Chu, Virginia; Prazeres, Duarte M F; Conde, Joao P

    2013-02-01

    The spatial and temporal control of biological species is essential in complex microfluidic biosystems. In addition, if the biological species is a cell, microfluidic handling must ensure that the cell's metabolic viability is maintained. The use of DEP for cell manipulation in microfluidics has many advantages because it is remote and fast, and the voltages required for cell trapping scale well with miniaturization. In this paper, the conditions for bacterial cell (Escherichia coli) trapping using a quadrupole electrode configuration in a PDMS microfluidic channel were developed both for stagnant and for in-flow fluidic situations. The effect of the electrical conductivity of the fluid, the applied electric field and frequency, and the fluid-flow velocity were studied. A dynamic exchange between captured and free-flowing cells during DEP trapping was demonstrated. The metabolic activity of trapped cells was confirmed by using E. coli cells genetically engineered to express green fluorescent protein under the control of an inducible promoter. Noninduced cells trapped by negative DEP and positive DEP were able to express green fluorescent protein minutes after the inducer was inserted in the microchannel system immediately after DEP trapping. Longer times of trapping prior to exposure to the inducer indicated first a degradation of the cell metabolic activity and finally cell death. PMID:23175163

  13. Interfacial microfluidic transport on micropatterned superhydrophobic textile.

    PubMed

    Xing, Siyuan; Jiang, Jia; Pan, Tingrui

    2013-05-21

    Textile-enabled interfacial microfluidics, utilizing fibrous hydrophilic yarns (e.g., cotton) to guide biological reagent flows, has been extended to various biochemical analyses recently. The restricted capillary-driving mechanism, however, persists as a major challenge for continuous and facilitated biofluidic transport. In this paper, we have first introduced a novel interfacial microfluidic transport principle to drive three-dimensional liquid flows on a micropatterned superhydrophobic textile (MST) platform in a more autonomous and controllable manner. Specifically, the MST system utilizes the surface tension-induced Laplace pressure to facilitate the liquid motion along the hydrophilic yarn, in addition to the capillarity present in the fibrous structure. The fabrication of MST is simply accomplished by stitching hydrophilic cotton yarn into a superhydrophobic fabric substrate (contact angle 140 ± 3°), from which well-controlled wetting patterns are established for interfacial microfluidic operations. The geometric configurations of the stitched micropatterns, e.g., the lengths and diameters of the yarn and bundled arrangement, can all influence the transport process, which is investigated both experimentally and theoretically. Two operation modes, discrete and continuous transport, are also presented in detail. In addition, the gravitational effect as well as the droplet removal process have been also considered and quantitatively analysed during the transport process. As a demonstration, an MST design has been implemented on an artificial skin surface to collect and remove sweat in a highly efficient and facilitated means. The results have illustrated that the novel interfacial transport on the textile platform can be potentially extended to a variety of biofluidic collection and removal applications.

  14. ParA-mediated plasmid partition driven by protein pattern self-organization

    PubMed Central

    Hwang, Ling Chin; Vecchiarelli, Anthony G; Han, Yong-Woon; Mizuuchi, Michiyo; Harada, Yoshie; Funnell, Barbara E; Mizuuchi, Kiyoshi

    2013-01-01

    DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low-copy plasmids, such as the plasmids P1 and F, employ a three-component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA, which also binds non-specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP-driven patterning is involved in partition is unknown. We reconstituted and visualized ParA-mediated plasmid partition inside a DNA-carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB-stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion-ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid. PMID:23443047

  15. Serum pattern profiling for analyzing different types of stress by protein chip technology: a preliminary study.

    PubMed

    Liu, Hui; Hou, Diandong; Wu, Da; Yin, Hong; Wu, Xiaoyi

    2010-01-01

    ProteinChip is a widely accepted tool for exploring serum pattern profile to evaluate the risk of somatic diseases from different stressors. In this study, by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-ToF), the serum proteome from mice under restraint and thermal stresses were profiled in detail and compared with the control group. Around 150 characteristic peaks were detected in all three groups, with m/z ranging from 1500 to 50,000, with most peaks being within the 2000 m/z to 20,000 m/z range. Compared with the control group, three significant protein peaks with m/z values of 2780, 3303 and 3450 appeared specifically in the restrained stress group and four other peaks with m/z values of 7500, 7811, 29,950 and 38,565 in the thermal stress group. Unexpectedly, no universal positive stress peaks were detected. These preliminary results clearly suggested that there might not be a common mechanism shared by various psychophysiological disorders under different stressors. By fast serum proteomics profiling, SELDI-ToF may be a convenient tool for evaluating the risk of stress-induced illness.

  16. Differentiating disease subtypes by using pathway patterns constructed from gene expressions and protein networks.

    PubMed

    Hung, Fei-Hung; Chiu, Hung-Wen

    2015-01-01

    Gene expression profiles differ in different diseases. Even if diseases are at the same stage, such diseases exhibit different gene expressions, not to mention the different subtypes at a single lesion site. Distinguishing different disease subtypes at a single lesion site is difficult. In early cases, subtypes were initially distinguished by doctors. Subsequently, further differences were found through pathological experiments. For example, a brain tumor can be classified according to its origin, its cell-type origin, or the tumor site. Because of the advancements in bioinformatics and the techniques for accumulating gene expressions, researchers can use gene expression data to classify disease subtypes. Because the operation of a biopathway is closely related to the disease mechanism, the application of gene expression profiles for clustering disease subtypes is insufficient. In this study, we collected gene expression data of healthy and four myelodysplastic syndrome subtypes and applied a method that integrated protein-protein interaction and gene expression data to identify different patterns of disease subtypes. We hope it is efficient for the classification of disease subtypes in adventure.

  17. Sec14-nodulin proteins and the patterning of phosphoinositide landmarks for developmental control of membrane morphogenesis

    PubMed Central

    Ghosh, Ratna; de Campos, Marília K. F.; Huang, Jin; Huh, Seong K.; Orlowski, Adam; Yang, Yuan; Tripathi, Ashutosh; Nile, Aaron; Lee, Hsin-Chieh; Dynowski, Marek; Schäfer, Helen; Róg, Tomasz; Lete, Marta G.; Ahyayauch, Hasna; Alonso, Alicia; Vattulainen, Ilpo; Igumenova, Tatyana I.; Schaaf, Gabriel; Bankaitis, Vytas A.

    2015-01-01

    Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it. Here we identify the conserved AtSfh1 Sec14-nodulin protein as a novel effector of phosphoinositide signaling in the extreme polarized membrane growth program exhibited by growing Arabidopsis root hairs. The data are consistent with Sec14-nodulin proteins controlling the lateral organization of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) landmarks for polarized membrane morphogenesis in plants. This patterning activity requires both the PtdIns(4,5)P2 binding and homo-oligomerization activities of the AtSfh1 nodulin domain and is an essential aspect of the polarity signaling program in root hairs. Finally, the data suggest a general principle for how the phosphoinositide signaling landscape is physically bit mapped so that eukaryotic cells are able to convert a membrane surface into a high-definition lipid-signaling screen. PMID:25739452

  18. Multiplex protein pattern unmixing using a non-linear variable-weighted support vector machine as optimized by a particle swarm optimization algorithm.

    PubMed

    Yang, Qin; Zou, Hong-Yan; Zhang, Yan; Tang, Li-Juan; Shen, Guo-Li; Jiang, Jian-Hui; Yu, Ru-Qin

    2016-01-15

    Most of the proteins locate more than one organelle in a cell. Unmixing the localization patterns of proteins is critical for u